Method of preparing basement membrane, method of constructing basement membrane specimen, reconstituted artificial tissue using the basement membrane specimen and process for producing the same

ABSTRACT

The object of the present invention is to provide the following: a method for preparing a basement membrane which is extracellular matrices having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells; a method for constructing a specimen of a basement membrane; a process for producing a reconstituted artificial tissue; a basement membrane specimen or an artificial tissue which can be transplanted while maintaining the structure of a basement membrane. A basement membrane having a barrier function is formed by culturing alveolar epithelial cells or vascular endothelial cells on a fibrous collagen matrix coated with a polymer having a sugar chain which can localize a receptor having an activity to accumulate a basement membrane component on the basal surface of the cells having an ability to form a basement membrane. A reconstructed artificial tissue is obtained by seeding and culturing desired homogeneous or heterogeneous cells having an ability to form a basement membrane on the basement membrane specimen constructed by the following process: the cells having an ability to form a basement membrane adhered onto a support structure through a basement membrane are treated with a surface active agent; the lipid component of cells is lysed; the mixture of an alkaline solution and a protease inhibitor is used to lyse the protein remained on the surface of the basement membrane of the cells. A protein support structure is temporarily adhered to plastic surface by hydrophobic bonding.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10,809,218filed on Mar. 25, 2004, now U.S. Pat. No. 7,399,634, which is acontinuation-in-part of International Patent Application PCT/JP02/09841filed Sep. 25, 2002 and published as WO 03/026712 on Apr. 3, 2003, whichclaims priority to Japanese Patent Application Numbers 2001-292510,2001-292675, 2001-292676, 2001-292677 and 2001-292510, all of which werefiled Sep. 25, 2001, and to Japanese Patent Application Numbers2002-278243 and 2002-278244, which were both filled on Sep. 24, 2002.Each of the above applications, and each document cited in this text andin each of the above applications (“application cited documents”) andeach document cited or referenced in each of the application citeddocuments, and any manufacturer's specifications or instructions for anyproducts mentioned in this text and in any document incorporated intothis text, are hereby incorporated herein by reference; and, technologyin each of the documents incorporated herein by reference can be used inthe practice of this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. Patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. Patent law; namely, that these terms are closed ended.

TECHNICAL FIELD

The present invention relates to: a method for preparing a basementmembrane which is extracellular matrices having a function to controlmorphology, differentiation, proliferation, motility, functionexpression and the like of cells; a tissue model which can be obtainedby the above-mentioned method for preparing a basement membrane; a testtissue kit including said tissue model (hereinafter referred to as “thepresent invention 1”). The present invention also relates to a methodfor constructing a basement membrane specimen, and a basement membranespecimen which can be obtained by said construction method (hereinafterreferred to as “the present invention 2”). The present invention furtherrelates to a reconstituted artificial tissue (reconstituted tissuemodel) of human and the like wherein a basement membrane specimen isused, for example, a reconstituted artificial tissue (reconstitutedtissue model) such as an artificial blood vessel, an artificial lung, anartificial liver, an artificial kidney, an artificial skin, anartificial cornea and the like, and a process for producing the same(hereinafter referred to as “the present invention 3”). The presentinvention still further relates to: a basement membrane specimen and anartificial tissue formed on a protein support structure which istemporarily adhered to plastic surface, more particularly to a basementmembrane specimen which is formed on a protein support attached toplastic surface but is temporarily adhered to the same in order to bephysically detached from plastic surface when desired; an artificialtissue (tissue model) such as an artificial blood vessel, an artificiallung, an artificial liver, an artificial kidney, an artificial skin, anartificial cornea and the like; a process for producing the same(hereinafter referred to as “the present invention 4”).

BACKGROUND ART

An epithelial tissue, which is a cell layer covering the inside andoutside surface of an animal body, such as an epidermis, a cornealepithelium, an alveolar epithelium, a mucosal epithelium of digestivesystem, renal glomerular epithelium, hepatic parenchymal cells and thelike, prevents the invasion of an exogenous material (microorganism,allergen, chemical substance, etc.) from the external world. The outerinterface of epithelial cells that institutes said epithelial tissue iscalled apical, and the inside undersurface is called basal. Just beneathsaid basal surface, there is a thin film structure of 50-100 nmthickness called a basement membrane comprised of extracellular matrices(ECM) such as proteins, proteoglycans and the like (not includingcells). A basement membrane is considered to be an essential structurefor immature epithelial cells to proliferate, to differentiate intomature cells, and to express its original morphology or function. Inother words, without a basement membrane, an epithelial tissue cannotmaintain itself or achieve its original performance. Although anepithelial cell layer of multilayer or monolayer prevents the invasionof an exogenous material from the external world as a barrier, abasement membrane itself also acts as a physical barrier. Thus,epithelial cells comprising an epithelial tissue collaborate with abasement membrane to form a solid barrier and to protect the internalvital activity.

A basement membrane, which is a specific membranous structure ofextracellular matrices formed on the interface of parenchymal cells,such as epithelial cells, endothelial cells, muscle cells, adipocytes,Schwann cells and the like, and connective tissue, is universally foundin respective tissue/organ of a living body, however, some basementmembranes (?) are highly specialized such as a renal glomerularcapillary loop, a nervous synapse membrane and the like. Therefore, notonly its function to adhere cells to an interstitium, but also itsfunction to selectively permeate a substance/cells, and to induce thedifferentiation of cells are also disclosed. In renal glomerulus,negative electric charge of a basement membrane is considered to beresponsible for the filtration function of kidney, and said negativeelectric charge is traditionally known to be charged by heparan sulfateproteoglycan (HSPG) which is currently called perlecan. HSPG is widelydistributed not only to a renal glomerular basement membrane but also tovarious basement membranes as its basic component in the same manner astype IV collagen, laminin, entactin and the like.

An extracellular matrix, especially abasement membrane, is now graduallyknown to be deeply involved not only in physiological phenomena such asgeneration or differentiation of an individual as mentioned above, butalso in formation of pathology such as proliferative metastasis ofcancer, inflammation and the like. Therefore, clarification of thefunction of its constituent protein has been an important task. Forexample, laminin, which is a main glycoprotein of a basement membrane,is a complex comprised of three subunits α, β, and γ, fifteen types ofits isoforms are known, and they are expressed tissue-specifically andat each step of development. Laminin is a complicated macromolecule of900,000 molecular weight having various bioactivities, and over 20 typesof laminin receptors are reported.

The interaction between a component of a basement membrane, which is athin extracellular matrix layer wherein cells can be adhered, andepithelial cells influences the cell function such as migration,proliferation, differentiation and the like (Crouch et al., Basementmembrane. In The Lung (ed. R. G. Crystal and J. B. West), pp 53.1-53.23.Philadelphia: Lippincott-Raven. 1996). As for the main components of abasement membrane, laminin, type IV collagen, heparan sulfateproteoglycan (HSPG), and entactin are known as mentioned above (Curr.Opin. Cell Biol. 6, 674-681, 1994), and mesenchymal cells are consideredto play an important role for the synthesis of a basement membranecomponent including isoform of laminin and type IV collagen (MatrixBiol. 14, 209-211, 1994; J. Biol. Chem. 268, 26033-26036, 1993),however, the role of epithelial cells is also important. HSPG isbelieved to have been derived from epithelial cells, however, laminin,type IV collagen, and entactin are synthesized in vivo by both ofepithelial cells and mesenchymal cells (Development 120, 2003-2014,1994; Gastroenterology 102, 1835-1845, 1992). Many attempts have beenmade to construct an epithelial tissue model in vitro showing acontinuous lamina densa. Tissue models of intestine (J. Cell Biol. 133,417-430, 1996) and skin (J. Invest. Dermatol. 105, 597-601, 1995; J.Invest. Dermatol. 109, 527-533, 1997; Dev. Dynam. 197. 255-267, 1993)and the like have been studied, and some of basement membrane componentsderived from mesenchymal cells have been found to play an important rolein the formation of a basement membrane.

Several methods to constitute a basement membrane by culturingepithelial cells, and to constitute epithelial cells wherein a basementmembrane structure is present just beneath the basal surface have beenreported. For example, the present inventors have reported that abasement membrane can be formed in vitro by coculturing alveolarepithelial cells and pulmonary fibroblasts (Cell Struc. Func., 22:603-614, 1997). It has been reported that: if pulmonary fibroblasts,being embedded in type I collagen gel, were cultured, the collagen gelwas contracted and became more solid by pulmonary fibroblasts, andextracellular matrices being secreted and adsorbed to collagen fiberaround the cells and deposited; such contracted collagen gel byfibroblasts is called a pseudointerstitium since it is similar to aninterstitium in vivo; and if type II alveolar epithelial cell lines(SV40-T2) were cultured on such a pseudointerstitial tissue forapproximately 14 days (T2-Fgel), basement membrane components such astype IV collagen, laminin and the like in extracellular matricessecreted by pulmonary fibroblasts were diffused in a culture medium andreached to the basal surface of the above-mentioned type II alveolarepithelial cell lines, and used as a material for the constitution of abasement membrane, and as a result, a basement membrane structure wasformed.

It is also reported that dilute neutral collagen solution was incubatedat 37° C. in 5% CO₂, and collagen fiber was formed, then air-driedcollagen fibrous matrix (fib) which was air-dried in aseptic conditionwas used as a alternative for the above-mentioned pseudointerstitium ina same manner as the above-mentioned coculture of alveolar epithelialcells and pulmonary fibroblasts to form a basement membrane (Eur. J.Cell Biol., 78:867-875, 1999; J. Cell Sci., 113:859-868, 2000). In thisprocess, if the concentration of collagen solution is high, there willbe less or no gap in fibrous collagen matrix formed, and if epithelialcells are cultured for a long term (10 days-2 weeks) for the purpose offorming a basement membrane, cells are detached and floated (e.g. BectonDickinson, Fibrous collagen coat culture insert), therefore, theconcentration of collagen solution is considered to be optimum at0.3-0.5 mg/ml (Eur. J. Cell. Biol., 78:867-875, 1999; J. Cell Sci.,113:859-868, 2000).

Type II alveolar epithelial cell lines (SV40-T2) were cultured onfibrous collagen matrix wherein Matrigel the registered trademark ofBecton Dickinson) was added, instead of using collagen matrix whereinfibrous cells were embedded. In this case, Matrigel functioned as anexogenous resource of basement membrane components. Matrigel is amixture of basement membrane components extracted fromEngelbreth-Holm-Swarm tumor matrix (J. Ekp. Med. 145, 204-220, 1977),and contains laminin-1, entactin, type IV collagen, and perlecan, aswell as various cytokines that possibly influence the ECM synthesis(Exp. Cell Res. 202, 1-8, 1992). In order to trace the Matrigelcomponents incorporated in a basement membrane, Matrigel was labeledwith biotin, and the process wherein the formation of a basementmembrane was accelerated depending on the amount of Matrigel, and abasement membrane matrix being secreted in punctiform manner depositedin a sheet form, then a basement membrane development was observed byimmunofluorescent staining of basement membrane components such aslaminin, entactin, type IV collagen, perlecan, and the electronmicrographic monitoring. As a result, it has been found that stableexogenous laminin-1 and entactin are largely involved in the completedevelopment of a basement membrane by the above-mentioned epithelialcells in vitro at the lower surface of alveolar epithelial cells (J.Cell Sci., 113:859-868, 2000).

Further, an artificial skin formation promoting agent and skin basementmembrane stabilizing agent comprising matrix metalloproteinase inhibitoror matrix metalloproteinase inhibitor and matrix protein productionpromoting agent; as well as a production method of artificial skincomprising adding matrix metalloproteinase inhibitor or matrixmetalloproteinase inhibitor and matrix protein production promotingagent to an artificial skin formation medium are known (JapaneseLaid-Open Patent Application No. 2001-269398).

The present inventors made a study on a process to form a basementmembrane structure just beneath the basal surface of epithelial cells ona fibrous collagen matrix by the coculture with fibroblasts-embeddedcollagen gel, and by the culture in the presence of TGF-β or Matrigel.In the case of type II alveolar epithelial cells, it was confirmed thata basement membrane was formed in the following cases as shown in FIG.1: the case wherein type II alveolar epithelial cells were cultured onpulmonary fibroblast-embedded collagen matrix in upper wells of cultureinserts (collagen gel wherein fibrous cells are embedded) (T2-Fgel); thecase wherein they were cultured on fibrous collagen substratum on upperwells in coculture with alveolar fibroblasts-embedded collagen matrix inlower wells (T2-fib-Fcm); the case wherein they were cultured on fibrouscollagen substratum in upper wells in the presence of Matrigel coat onlower wells (T2-fib-MG); the case wherein they are cultured on fibrouscollagen substratum in upper wells in the presence of growth factorTGF-β in upper and lower wells (T2-fib-TGFβ). However, it was alsoconfirmed that type II alveolar epithelial cells were unable toconstitute a basement membrane without the supply of, for example, afibroblast, exogenous basement membrane components from Matrigel or agrowth factor TGF-β, since the endogenous basement membrane componentsfrom type II alveolar epithelial cells are not enough for the cells toassemble a basement membrane structure effectively. However, followingproblems still remained: a problem of frequent troubles that, whenfibroblasts secreting basement membrane components and a growth factorof TGF-β are used, gel contraction occurs during the culture, and thefibroblasts are peeled off from plastic membrane together with alveolarepithelial cells; a problem that reagents used to peel alveolarepithelial cells for the preparation of basement membrane specimen tendto remain inside the gel if fibroblasts are embedded, and the washprocedure of such reagents is complicated; a problem that if some partsof cells remain, it may become an antigen; a problem that cultureprotocol for the formation of a basement membrane itself is complicated.Further, there has been also a problem that if fibroblasts alternativesuch as Matrigel and the like or a growth factor TGF-β is used as aresource of basement membrane component, said Matrigel and said growthfactor TGFβ are expensive, and it is not advantageous in terms of thecost.

On the other hand, constitution of a basement membrane by endothelialcells (EC) was also considered. Although a basement membrane which ispresent just beneath the basal surface of endothelial cells alsocontributes to the expression and the maintenance of functions inendothelial cells, and a basement membrane of endothelial cells plays arole of a barrier when inflammatory cells invade into tissue from bloodvessel, or when cancer cells metastasize, a basement membrane ofvascular endothelial cells cannot be easily formed as for the case ofepithelial cells. In the formation of a basement membrane by vascularendothelial cells, unlike the case of type II alveolar epithelial cells,as shown in FIG. 2, a basement membrane was not formed in the followingcases with the exception of the case of (EC-Fgel) wherein the culturewas carried out on fibroblast-embedded collagen matrix in upper wells;the case wherein the culture was carried out on fibrous collagensubstratum in (on) upper wells in the presence of pulmonaryfibroblasts-embedded collagen matrix in lower wells (EC-fib-Fcm); thecase wherein the culture was carried out on fibrous collagen substratumin upper wells in the presence of Matrigel coat in lower wells(EC-fib-MG); the case wherein the culture was carried out on fibrouscollagen matrix in upper wells (EC-fib).

In the meantime, the present inventors have reported that epithelialcells can be automatically detached from abasement membrane if alveolarepithelial cells which formed the above-mentioned basement membrane aretreated with 0.18 M of hydrogen peroxide solution for 10 minutes,continued to culture for an additional day (Cell Struc. Func., 22,603-614, 1997). In such process, however, it has been found to be aproblem that there are cases that artificial human tissue havingsufficient physiological activity such as function expression andmaintenance of cells even if homogeneous or heterogeneous cells having acertain ability to form a basement membrane are seeded and cultured onsaid basement membrane since there are some cases wherein the detachmentof cells from a basement membrane is insufficient, and a part of abasement membrane is damaged.

The object of the present invention 1 is to provide: a method forpreparing a basement membrane which is extracellular matrices having afunction to control morphology, differentiation, proliferation,motility, function expression and the like of cells, particularly amethod for preparing a basement membrane with which cells having anability to form a basement membrane such as epithelial cells,endothelial cells and the like can effectively activate an endogenousbasement membrane component; a tissue model which can be obtained by theabove-mentioned method for preparing a basement membrane; a test tissuekit including said tissue model. The object of the present invention 2is to easily and in the short term provide a basement membrane specimenhaving a function to control morphology, differentiation, proliferation,motility, function expression and the like of cells when a certainhomogeneous or heterogeneous cells having an ability to form a basementmembrane are seeded and cultured. The object of the present invention 3is to provide a process for producing a reconstituted artificial tissuehaving versatility wherein desired artificial tissue can be producedeasily and efficiently in the short term at any time and any place whenneeded by seeding and culturing certain cells which are homogeneous orheterogeneous to cells which formed a basement membrane using a basementmembrane specimen having a function to control morphology,differentiation, proliferation, motility, function expression and thelike of cells as a common base material for tissue construction. Anotherobject of the present invention 3 is to provide a reconstitutedartificial tissue such as a tissue model, an organ model and the like,which can be obtained by said process for producing an artificialtissue, which has cell layers and a basement membrane structure withbarrier function original to a living body, and which can beadvantageously applied to pharmacological test, toxicity test or thelike of chemical substances. The object of the present invention 4 is toprovide a basement membrane specimen which is extracellular matriceshaving a function to control morphology, differentiation, proliferation,motility, function expression and the like of cells, with much higherversatility since it is possible to transplant while maintaining thestructure of a basement membrane, and which is formed on a proteinsupport structure which is temporarily adhered to plastic surface inorder to be physically detached from plastic surface when needed whileit is adsorbed and fixed on plastic surface when a basement membrane andan artificial tissue are prepared. The examples include: a basementmembrane specimen formed on a collagen fiber; an artificial tissue andan artificial organ such as an artificial blood vessel, an artificiallung, an artificial liver, an artificial kidney, an artificial skin, anartificial cornea and the like; for example, an artificial tissue on acollagen fiber which formed a basement membrane structure on a matrixjust beneath the cells.

Basement membrane components secreted from the cells having an abilityto be assembled into a basement membrane by such as epithelial cells,endothelial cells and the like, or from fibroblasts, cannotautomatically form a basement membrane structure by themselves, andneeds a receptor which is considered to be localized on the surface ofthe cells having an ability to form a basement membrane such asepithelial cells, endothelial cells and the like, or particularly on thebasal surface of said cells. The identity of said receptor, however, isnot clearly known at the moment including the concern whether it is asingle protein. The present inventors, in the course of a keen study onthe mechanism of a basement membrane formation, obtained the knowledgethat type II alveolar epithelial cells or vascular endothelial cellsexpress receptors for sugar chains on their basal surface since type IIalveolar epithelial cells or vascular endothelial cells can be adheredto a polymer having a certain sugar chain in vitro, namely a sugar chainwhich can localize a receptor having an activity to accumulate abasement membrane component on the basal surface of the cells having anability to form a basement membrane, e.g. a sugar-chain coat havingβ-D-glucopyranosyl nonreducing end or2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end. Secondly, as aresult of culturing type II alveolar epithelial cells or vascularendothelial cells on fibrous collagen substratum coated with theabove-mentioned polymer, the present inventors have found that abasement membrane having a barrier function similar to the one seen invivo is formed just beneath the type II alveolar epithelial cells orvascular endothelial cells. It was also found that although the supplyof a basement membrane component such as a Matrigel and the like, andthe addition of TGF-β was not necessary for the formation of saidbasement membrane, if Matrigel was added, construction of a basementmembrane was significantly accelerated, the culture term was long enoughwith a week, and the basement membrane became several fold thick. Theknowledge that the above-mentioned fact is a result of that the receptoragainst the above-mentioned sugar chain was involved to progress thebasement membrane formation was obtained. The present invention 1 hasbeen completed based on such knowledge.

DISCLOSURE OF THE INVENTION

The present invention relates to: a method for preparing a basementmembrane wherein cells having an ability to form a basement membrane arecultured on a support structure with a sugar-chain coat which canlocalize a receptor having an activity to accumulate basement membranecomponents onto a basal surface of the cells having an ability to form abasement membrane (paragraph 1); the method for preparing a basementmembrane according to paragraph 1, wherein the cells having an abilityto form a basement membrane are cultured on of a support structure withboth opposite surfaces coated by a sugar chain (paragraph 2); the methodfor preparing a basement membrane according to paragraph 1 or 2, whereina component secreted from the cells having an ability to form a basementmembrane is used as a basement membrane component (paragraph 3); themethod for preparing a basement membrane according to any of paragraphs1-3, wherein a sugar-chain coat, which can possibly adhere the cellshaving an ability to form a basement membrane onto a support structurethrough the binding between a sugar chain or a part of a sugar chain anda receptor, is used (paragraph 4); the method for preparing a basementmembrane according to paragraph 4, wherein a sugar-chain coat is used,the sugar chain or a part of the sugar chain that binds to a receptorcan be replaced by a basement membrane component (paragraph 5); themethod for preparing a basement membrane according to any of paragraphs1-5, wherein the support structure with a sugar-chain coat is a supportstructure coated with a polymer having a sugar chain (paragraph 6); themethod for preparing a basement membrane according to paragraph 6,wherein the polymer having a sugar chain is a polymer having a sugarchain with β-D-glucopyranosyl nonreducing end or2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end (paragraph 7);the method for preparing a basement membrane according to paragraph 7,wherein one or more types of polymers selected from PV-GlcNAc, PV-CA andPV-Lam is used as the polymer having a sugar chain (paragraph 8).

The present invention is also related to: the method for preparing abasement membrane according to any of paragraphs 1-8, wherein the cellshaving an ability to form a basement membrane are cocultured withfibroblasts or their alternatives (paragraph 9); the method forpreparing a basement membrane according to any of paragraphs 1-9,wherein the cells having an ability to form a basement membrane arecultured in the presence of one or more types of basement membranecomponents (paragraph 10); the method for preparing a basement membraneaccording to any of paragraphs 1-10, wherein the cells having an abilityto form a basement membrane are cultured in the presence of TGF-β(transforming growth factor) (paragraph 11); the method for preparing abasement membrane according to any of paragraphs 1-11, wherein the cellshaving an ability to form a basement membrane are epithelial cells,endothelial cells or mesenchymal cells (paragraph 12); the method forpreparing a basement membrane according to any of paragraphs 1-12,wherein the cells and/or fibroblasts having an ability to form abasement membrane are basement membrane component-hyperexpressing cellsinto which genes of one or more types of a basement membrane componentare transfected (paragraph 13); the method for preparing a basementmembrane according to any of paragraphs 1-13, wherein the supportstructure is a fibrous collagen (paragraph 14); a tissue model which canbe obtained by the method for preparing a basement membrane according toany of paragraphs 1-14 (paragraph 15); a test tissue kit including atissue model which can be obtained by the method for preparing abasement membrane according to any of paragraphs 1-14 (paragraph 16).

The present inventors found the following facts: if an surface activeagent e.g. 0.1% Triton X-100 (Calbiochem-Novabiochem Corporation) isused for the cells wherein the basement membrane obtained by theabove-mentioned present invention 1 is formed, lipid component of thecells is lysed by its surface activity; if an alkaline solution e.g.10-50 mM of NH₃ is used, protein residues remained on the basementmembrane of cells is lysed; if protease inhibitors cocktail (PIC) isused, degradation of a basement membrane by endogenous proteases and thelike in lysosomes being liberated when cells are lysed is suppressed; ifdesired homogeneous or heterogeneous cells having an ability to form abasement membrane are seeded and cultured, a basement membrane specimenhaving a function to control morphology, differentiation, proliferation,motility, function expression and the like of cells can be obtained inthe short term. Thus, the present invention 2 has been completed.

The present invention 2 relates to: a method for constructing a basementmembrane specimen wherein cells having an ability to form a basementmembrane adhered onto a support structure through a basement membraneare removed using a solvent having the ability to lyse lipid of thecells and an alkaline solution (paragraph 17); the method forconstructing a basement membrane specimen according to paragraph 17,wherein the treatment to remove proteinous and nucleic residues using analkaline solution is conducted after or at the same time as thedelipidating treatment using a solvent having the ability to lyse lipidis conducted (paragraph 18); the method for constructing a basementmembrane specimen according to paragraph 17 or 18, wherein the solventhaving the ability to lyse lipid is a surface active agent (paragraph19); the method for constructing a basement membrane specimen accordingto paragraph 19, wherein the surface active agent is Triton X-100(paragraph 20); the method for constructing a basement membrane specimenaccording to any of paragraphs 17-20, wherein the alkaline solution isan alkaline solution with pH 8-14 (paragraph 21); the method forconstructing a basement membrane specimen according to paragraph 21,wherein the alkaline solution is an alkaline solution with pH 9-10(paragraph 22); the method for constructing a basement membrane specimenaccording to any of paragraphs 17-22, wherein a protease inhibitor isfurther used (paragraph 23).

The present invention 2 also relates to: the method for constructing abasement membrane specimen according to any of paragraphs 17-23, whereinthe basement membrane is a basement membrane prepared by culturing thecells having an ability to form a basement membrane on a collagen gelwherein fibroblasts are embedded (paragraph 24); the method forconstructing abasement membrane specimen according to any of paragraphs17-24, wherein the basement membrane is a basement membrane prepared byculturing the cells having an ability to form a basement membrane on asupport structure with a sugar-chain coat which can localize a receptorhaving an activity to accumulate a basement membrane component on thebasal surface of the cells having an ability to form a basement membrane(paragraph 25); the method for constructing a basement membrane specimenaccording to any of paragraphs 17-25, wherein the basement membrane is abasement membrane prepared by culturing the cells having an ability toform a basement membrane in the presence of a matrix metalloproteinase(paragraph 26); the method for constructing a basement membrane specimenaccording to any of paragraphs 17-26, wherein the basement membrane is abasement membrane prepared by culturing a basement membrane componentand/or growth factor hyperexpressing cells into which one or more typesof genes and/or growth factors of a basement membrane component areintroduced (paragraph 27); a basement membrane specimen which can beobtained by the method for constructing a basement membrane specimenaccording to any of paragraphs 17-27 (paragraph 28); the basementmembrane specimen according to paragraph 28, which is detached from asupport structure (paragraph 29).

The present inventors also have found that as a result of seeding andculturing desired homogeneous or heterogeneous cells having an abilityto form a basement membrane on the basement membrane specimen which canbe obtained by the above-mentioned present invention 2, an artificialtissue having a barrier function original to a living body can beconstituted.

Thus, the present invention 3 relates to: a process for producing areconstituted artificial tissue wherein certain cells having an abilityto form a basement membrane are seeded and cultured on a basementmembrane specimen or amorphously basement membrane components-depositedspecimen (paragraph 30); the process for producing a reconstitutedartificial tissue according to paragraph 30, wherein the cells having anability to form a basement membrane have a different origin from that ofa basement membrane specimen or amorphously basement membranecomponents-deposited specimen (paragraph 31); the process for producinga reconstituted artificial tissue according to paragraph 30 or 31,wherein the basement membrane specimen or the amorphously basementmembrane components-deposited specimen is obtained by removing the cellshaving an ability to form a basement membrane which are adhered onto asupport structure through a basement membrane or basement membranecomponents-amorphous deposits using a solvent having the ability to lyselipid of the cells and an alkaline solution (paragraph 32); the processfor producing a reconstituted artificial tissue according to paragraph32, wherein the solvent having the ability to lyse lipid of cells is asurface active agent (paragraph 33); the process for producing areconstituted artificial tissue according to paragraph 32 or 33, whereinthe alkaline solution is an alkaline solution with pH 8-14 (paragraph34); the process for producing a reconstituted artificial tissueaccording to paragraph 34, wherein the alkaline solution is an alkalinesolution with pH 9-10 (paragraph 35); the process for producing areconstituted artificial tissue according to any of paragraphs 32-35,wherein a protease inhibitor is further used (paragraph 36); the processfor producing a reconstituted artificial tissue according to any ofparagraphs 30-36, wherein the basement membrane specimen or the basementmembrane components-amorphous deposits specimen is obtained from abasement membrane or a basement membrane components-amorphous depositsprepared by culturing the cells having an ability to form a basementmembrane on a collagen gel wherein fibroblasts are embedded (paragraph37); the process for producing a reconstituted artificial tissueaccording to any of paragraphs 30-37, wherein the basement membranespecimen or the basement membrane components-amorphous deposits specimenis obtained from a basement membrane or a basement membranecomponents-amorphous deposits prepared by culturing the cells having anability to form a basement membrane on a support structure with a sugarchain-coat which can localize a receptor having an activity toaccumulate a basement membrane component onto the basal surface of thecells having an ability to form a basement membrane or the surface ofthe basement membrane components amorphous deposits (paragraph 38) theprocess for producing a reconstituted artificial tissue according to anyof paragraphs 30-38, wherein the basement membrane specimen or thebasement membrane components-amorphous deposits specimen is obtainedfrom a basement membrane or a basement membrane components-amorphousdeposits prepared by culturing the cells having an ability to form abasement membrane in the presence of a matrix metalloproteinase(paragraph 39); the process for producing a reconstituted artificialtissue according to any of paragraphs 30-39, wherein the basementmembrane specimen or the basement membrane components-amorphous depositsspecimen is obtained from a basement membrane or a basement membranecomponents-amorphous deposit prepared by culturing a basement membranecomponent and/or growth factor hyperexpressing cells into which one ormore types of genes and/or growth factors of basement membranecomponents are transfected (paragraph 40).

The present invention also relates to: a reconstituted artificial tissuewhich can be obtained by the production process according to any ofparagraphs 30-40 paragraph 41); the reconstituted artificial tissueaccording to paragraph 41, wherein the reconstituted artificial tissueis an artificial blood vessel, an artificial lung, an artificial liver,an artificial kidney, an artificial skin or an artificial cornea(paragraph 42); the reconstituted artificial tissue according toparagraph 41 or 42, wherein the reconstituted artificial tissue is anartificial human tissue (paragraph 43); the reconstituted artificialtissue according to any of paragraphs 41-43, which is detached from asupport structure (paragraph 44); a method for testing the safety andtoxicity of a test substance wherein the reconstituted artificial tissueaccording to any of paragraphs 41-44 is used (paragraph 45).

The artificial tissue prepared by the present invention 3 has a barrierfunction original to the living body since it has cell layers and abasement membrane structure with a barrier function original to theliving body, and can be advantageously applied as a tissue model to apharmacological test, toxicity test and the like for chemical products.However, a support structure supporting an artificial tissue and abasement membrane specimen is formed in the condition of adhering toplastic surface, therefore it cannot be used as a regenerative medicalmaterial in such adhering condition, and its basement membrane structurewill be broken if such a support structure is mechanically detached fromplastic surface and it cannot be used as a tissue having physiologicalactivity such as a barrier function original to the living body. It was,however, found that if a protein support structure is temporarilyadhered to plastic surface through an adsorptive polymer by hydrophobicbonding having a hydrophobic linear carbon skeleton and a functionalgroup which can react with protein in a molecule such as an alternatingcopolymer of methyl vinyl ether and maleic anhydride, and then anartificial tissue or a basement membrane specimen is formed on such aprotein support structure supporting an artificial tissue or a basementmembrane specimen can be physically detached from plastic surface whendesired, and such a protein support structure supporting the detachedartificial tissue and basement membrane specimen can be transplantedwhile maintaining the basement membrane structure. Thus the presentinvention 4 has been completed.

The present invention 4 relates to: a basement membrane specimen or anartificial tissue which is formed on a protein support structuretemporarily adhered to plastic surface through an adsorptive polymer byhydrophobic bonding having a hydrophobic linear carbon skeleton and afunctional group which can react with protein in a molecule (paragraph46); the basement membrane specimen or the artificial tissue accordingto paragraph 46, wherein the adsorptive polymer by hydrophobic bondingis an adsorptive polymer by hydrophobic bonding shown by the followinggeneral formula [I] (paragraph 47):

(In the formula, X denotes CH or NHCHCO, Y denotes CH or NHCR²CO, R¹denotes H, alkyl group of C1-C3, alkoxy group of C1-C3 or aryl group ofC6-C8, R² denotes H or alkyl group of C1-C3, Z denotes a functionalgroup (reactional group) optionally bonded to each other, spacer denotes(—CH₂-)p or (—NHCHR³HCO-)q, R³ denotes H or alkyl group of C1-C3, mdenotes an integral number greater or equal to 1, n denotes an integralnumber between 100 and 20000, p and q independently denote 0 or integralnumbers 1-8, r denotes an integral number greater or equal to 1); thebasement membrane specimen or the artificial tissue according toparagraph 47, wherein the adsorptive polymer by hydrophobic bondingshown by the general formula [I] is alternating copolymer of methylvinyl ether and maleic anhydride (paragraph 48); the basement membranespecimen or the artificial tissue according to any of paragraphs 46-48,wherein the basement membrane specimen is a basement membrane specimenconstructed by removing the cells having an ability to form a basementmembrane adhered onto a protein support structure through a basementmembrane using a solvent having the ability to lyse lipid of the cellsand an alkaline solution (paragraph 49); the basement membrane specimenor the artificial tissue according to any of paragraphs 46-49, whereinthe artificial tissue is artificial tissue prepared by culturing thecells having an ability to form a basement membrane on a protein supportstructure (paragraph 50); the basement membrane specimen or theartificial tissue according to any of paragraphs 46-50, wherein theartificial tissue is an artificial tissue prepared by culturing thecells having an ability to form a basement membrane on a protein supportstructure with a sugar-chain coat which can localize a receptor havingan activity to accumulate a basement membrane component onto the basalsurface of the cells having an ability to form a basement membrane(paragraph 51); the basement membrane specimen or the artificial tissueaccording to any of paragraphs 46-51, wherein the protein supportstructure is a collagen gel wherein fibroblasts are embedded (paragraph52); the basement membrane specimen or the artificial tissue accordingto any of paragraphs 46-49, wherein the artificial tissue is anartificial tissue prepared by culturing the cells having an ability toform a basement membrane in the presence of a matrix metalloproteinase(paragraph 53); the basement membrane specimen or the artificial tissueaccording to any of paragraphs 46-53, wherein the artificial tissue isan artificial tissue prepared by culturing a basement membrane componentand/or growth factor hyperexpressing cells into which one or more typesof genes and/or growth factors of basement membrane components aretransfected (paragraph 54); the basement membrane specimen or theartificial tissue according to any of paragraphs 46-49, wherein theartificial tissue is a reconstituted artificial tissue prepared byseeding and culturing the cells having certain ability to form abasement membrane on a basement membrane specimen paragraph 55); thebasement membrane specimen or the artificial tissue according to any ofparagraphs 46-55, wherein the cells having an ability to form a basementmembrane are epithelial cells or endothelial cells (paragraph 56); thebasement membrane specimen or the artificial tissue according to any ofparagraphs 46-56, wherein the artificial tissue is an artificialepidermal tissue, an artificial corneal epithelial tissue, an artificialalveolar epithelial tissue, an artificial respiratory epithelial tissue,an artificial renal glomerular tissue, an artificial hepatic parenchymaltissue or an artificial pulmonary arterial vascular endothelial tissue,or, an artificial blood vessel, an artificial lung, an artificial liver,an artificial kidney, an artificial skin or an artificial cornea(paragraph 57).

The present invention 4 also relates to: a process for producing abasement membrane specimen or an artificial tissue which can betransplanted while maintaining the structure of a basement membranewherein a protein support structure is temporarily adhered to plasticsurface through an adsorptive polymer by hydrophobic bonding having ahydrophobic linear carbon skeleton and a functional group which canreact with protein in a molecule, and a basement membrane specimen or anartificial tissue is formed thereon, and a protein support structuresupporting a basement membrane specimen or an artificial tissue isphysically detached from plastic surface when desired (paragraph 58);the process for producing a basement membrane specimen or an artificialtissue which can be transplanted while maintaining the structure of abasement membrane according to paragraph 58, wherein the adsorptivepolymer by hydrophobic bonding is an adsorptive polymer by hydrophobicbonding shown by the following general formula [I] (paragraph 59):

(In the formula, X denotes CH or NHCHCO, Y denotes CH or NHCR²CO, R¹denotes H, alkyl group of C1-C3, alkoxy group of C1-C3 or aryl group ofC6-C8, R² denotes H or alkyl group of C1-C3, Z denotes a functionalgroup (reactional group) optionally bonded to each other, spacer denotes(—CH₂-)p or (—NHCHR³HCO-)q, R³ denotes H or alkyl group of C1-C3, mdenotes an integral number greater or equal to 1, n denotes an integralnumber between 100 and 20000, p and q independently denote 0 or integralnumbers 1-8, r denotes an integral number greater or equal to 1); theprocess for producing a basement membrane specimen or an artificialtissue which can be transplanted while maintaining the structure of abasement membrane according to paragraph 59, wherein the adsorptivepolymer by hydrophobic bonding shown by the general formula [I] is analternating copolymer of methyl vinyl ether and maleic anhydride(paragraph 60); the process for producing a basement membrane specimenor an artificial tissue which can be transplanted while maintaining thestructure of a basement membrane according to any of paragraphs 58-60,wherein the basement membrane specimen is a basement membrane specimenconstructed by removing the cells having an ability to form a basementmembrane adhered onto a protein support structure through a basementmembrane using a solvent having the ability to lyse lipid of the cellsand an alkaline solution (paragraph 61); the process for producing abasement membrane specimen or an artificial tissue which can betransplanted while maintaining the structure of a basement membraneaccording to any of paragraphs 58-61, wherein the artificial tissue is abasement membrane prepared by culturing the cells having an ability toform a basement membrane on a protein support structure (paragraph 62);the process for producing a basement membrane specimen or an artificialtissue which can be transplanted while maintaining the structure of abasement membrane according to any of paragraphs 58-62, wherein theartificial tissue is an artificial tissue prepared by culturing thecells having an ability to form a basement membrane on a protein supportstructure with a sugar-chain coat which can localize a receptor havingan activity to accumulate a basement membrane component onto the basalsurface of the cells having an ability to form a basement membrane(paragraph 63); the process for producing a basement membrane specimenor an artificial tissue which can be transplanted while maintaining thestructure of a basement membrane according to any of paragraphs 58-63,wherein the artificial tissue is a reconstituted artificial tissueprepared by seeding and culturing the cells having a certain ability toform a basement membrane on the basement membrane specimen (paragraph64); the process for producing a basement membrane specimen or anartificial tissue which can be transplanted while maintaining thestructure of a basement membrane according to any of paragraphs 58-64,wherein the cells having an ability to form a basement membrane areepithelial cells or endothelial cells (paragraph 65); the process forproducing a basement membrane specimen or an artificial tissue which canbe transplanted while maintaining the structure of a basement membraneaccording to any of paragraphs 58-65, wherein the protein supportstructure is a collagen gel wherein fibroblasts are embedded (paragraph66); the process for producing the basement membrane specimen or theartificial tissue which can be transplanted while maintaining thestructure of a basement membrane according to any of paragraphs 58-66,wherein the artificial tissue is an artificial epidermal tissue, anartificial corneal epithelial tissue, an artificial alveolar epithelialtissue, an artificial respiratory epithelial tissue, an artificial renalglomerular tissue, an artificial hepatic parenchymal tissue or anartificial pulmonary arterial vascular endothelial tissue, or, anartificial blood vessel, an artificial lung, an artificial liver, anartificial kidney, an artificial skin or an artificial cornea (paragraph67).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a set of schematic diagrams showing the formation of abasement membrane by alveolar epithelial cells.

FIG. 2 is a set of schematic diagrams showing the formation of abasement membrane by pulmonary arterial vascular endothelial cells.

FIG. 3 is a set of schematic diagrams showing the formation of abasement membrane by the coculture of epithelial cells and endothelialcells.

FIG. 4 is a set of schematic diagrams showing the construction of abasement membrane and the constitution of an alveolar epithelial tissue.

FIG. 5 is a set of schematic diagrams showing the constitution of anendothelial tissue and an epithelial tissue using a reconstitutedbasement membrane specimen.

FIG. 6 is a drawing showing the adhesion specificity of type II alveolarcells on sugar chains.

FIG. 7 is a set of transmission electron micrographs of an alveolarepithelial tissue formed as a result of 2-week culture of type IIalveolar epithelial cells on the high density collagen fiber (fib*)coated with various kinds of PV-sugars.

FIG. 8 is a set of scanning electron micrographs of extracellularmatrices beneath an alveolar epithelial tissue formed as a result of2-week culture of type II alveolar epithelial cells on the high densitycollagen fiber (fib*) coated with various kinds of PV-sugars.

FIG. 9 is a set of transmission electron micrographs of an alveolarepithelial tissue (left) and scanning electron micrographs ofextracellular matrices beneath an alveolar epithelial tissue (right),both of which were formed as a result of 10-day coculture of type IIalveolar epithelial cells with 25 μl of Matrigel coat on the bottom faceof a culture dish on the high density collagen fiber (fib*) coated withvarious kinds of PV-sugars.

FIG. 10 is a set of scanning electron micrographs of extracellularmatrices which show the result of basement membrane formation of humanpulmonary arterial vascular endothelial cells by the culture methodshown in FIG. 2.

FIG. 11 is a set of scanning electron micrographs of extracellularmatrices beneath vascular endothelial cell layers formed as a result ofseeding human alveolar artery vascular endothelial cells on the highdensity collagen fiber (fib*) coated with various kinds of PV-sugars andcoculturing for 2 weeks with a collagen gel wherein alveolar fibroblastsare embedded (EC-fib*-Fcm).

FIG. 12 is a set of transmission electron micrographs of a respiratoryepithelial tissue constituted on a reconstructed basement membranespecimen.

FIG. 13 is a set of transmission electron micrographs of a humanvascular endothelial tissue constituted on a reconstructed basementmembrane specimen.

FIG. 14 is a set of scanning electron micrographs of collagen fiber(fib) (A), amorphous deposits structure (deposit-fib: shown with *) (B)and a reconstructed basement membrane specimen (rBM) (C) whereinsecreted deposits beneath the cells is exposed to the surface as aresult of removing the cells after the culture of alveolar epithelialcells on a collagen fiber under the condition that a basement membraneis not formed and that a basement membrane is formed, and transmissionelectron micrographs (D-F) of type II alveolar epithelial tissuereconstituted thereon.

FIG. 15 is a set of transmission electron micrographs (A-C) of arespiratory epithelial tissue reconstituted on collagen fiber (fib),amorphous deposit structure (deposit-fib), and a basement membranespecimen (rBM).

FIG. 16 is a set of transmission electron micrographs of type IIalveolar epithelial tissue constituted on collagen fiber (fib) in thepresence of synthetic inhibitor of matrix metalloproteinase (MMP).

BEST MODE OF CARRYING OUT THE INVENTION

As for the method for preparing a basement membrane of the presentinvention 1, there is no particular limitation as long as it is a methodwherein the cells having an ability to form a basement membrane arecultured on a protein support structure with a specific sugar-chaincoat, namely a sugar chain which can localize a receptor having anactivity to accumulate a basement membrane component on the basalsurface of the cells having an ability to form a basement membrane. Theabove-mentioned cells having an ability to form a basement membrane canbe exemplified by epithelial cells, endothelial cells, mesenchymal cellsand the like. The above-mentioned epithelial cells can be moreparticularly exemplified by epidermal cells, corneal epithelial cells,alveolar epithelial cells, mucosal epithelial cells of digestive organsystem, renal glomerular epithelial cells, hepatic parenchymal cells andthe like, the above-mentioned endothelial cells can be more particularlyexemplified by renal glomerular capillary endothelial cells, pulmonaryarterial vascular endothelial cells, placental venous vascularendothelial cells, or aortic endothelial cells and the like, and themesenchymal cells can be more particularly exemplified by muscle cells,adipocytes, glial cells, Schwann cells and the like.

A basement membrane component such as laminin, type IV collagen, heparansulfate proteoglycan (HSPG), entactin or the like is required for thepreparation of a basement membrane, and respective cells having anability to form a basement membrane secret basement membrane components.The basement membrane components secreted from such cells, however, aresecreted from the basal surface (lower surface) of the cells toward theinside of extracellular matrices formed by fibrous collagen matrix.Therefore, most of the secreted basement membrane components are a partfrom the basal surface, and they diffuse from the basal surface into aculture medium, or they are degraded by proteases in mid course, as aresult, usually they are not effectively utilized. However, in themethod for preparing a basement membrane of the present invention 1, anendogenous basement membrane component secreted from cells having anability to form a basement membrane such as the above-mentionedepithelial cells, endothelial cells and the like can be more effectivelyutilized by culturing the cells having an ability to form a basementmembrane on a protein support structure with a specific sugar-chaincoat, namely a sugar chain which can localize a receptor having anactivity to accumulate a basement membrane component to the basalsurface of the cells having an ability to form a basement membrane.

Further, if the method for present invention 1 for culturing the cellshaving an ability to form a basement membrane is conducted on the twoopposite basal surface of the protein support structure with sugar-chaincoat, for example, if fibrous collagen is generated on the both sides ofporous membrane, two types of cells having an ability to form a basementmembrane such as the combination of epithelial cells and vascularendothelial cells and the like are seeded and cultured on its bothsides, the diffusion of endogenous basement membrane components secretedfrom the cells having an ability to form a basement membrane isprevented, so that effective utilization of basement membrane componentscan be enhanced. In other words, the basement membrane componentssecreted from the cells on one side reaches to the other cellspositioned on the opposite side of fibrous collagen, and is blocked bythe barrier without a gap due to the cell-cell junction (tight junction)formed by such cells, and do not diffuse into culture medium, and as aresult, the effective utilization of basement membrane components can beenhanced. The upper stand of FIG. 3 schematically shows the following:the formation of a basement membrane by the coculture of epithelialcells and vascular endothelial cells through collagen fiber in thepresence of fibroblasts embedded in collagen gel (left); the formationof a basement membrane by the coculture of epithelial cells and vascularendothelial cells through a thin membrane of collagen fiber which is asupport structure with the sugar-chain coat of the present invention(middle); the formation of a basement membrane by the coculture ofepithelial cells and fibroblasts through collagen fiber (right).Further, the lower stand of FIG. 3 shows the conditions wherein the celltissues which are not needed such as a vascular endothelial tissue(left), epithelial tissue (middle), fibroblasts (right) are mechanicallyexfoliated. As for the combination of these cells, the followingcombinations are considered: epithelial cells and vascular endothelialcells; epithelial cells and epithelial cells; endothelial cells andendothelial cells; epithelial cells or endothelial cells and somemesenchymal cells, etc. The above-mentioned support structure can beexemplified by porous PET membrane, elastin (polymer) membrane, as wellas fibrous collagen membrane, fibrous collagen matrix.

Further, in the method for preparing a basement membrane of the presentinvention 1, in order that a basement membrane can be prepared in theshort term using even exogenous basement membrane components in additionto endogenous basement membrane components secreted from these cellshaving an ability to form a basement membrane, it is also possible tococulture with fibroblasts secreting basement membrane components andTGF-β, or more preferably with conditioned culture medium of fibroblastsor a fibroblast substitute such as Matrigel richly containing basementmembrane components. Besides, in order that a basement membrane an beprepared in the short term in the same manner, it is also possible toculture the cells having an ability to form a basement membrane in thepresence of one or more types of basement membrane components separatelyprepared such as laminin, type IV collagen, heparan sulfate proteoglycan(HSPG), entactin and the like, or in the presence of TGF-β. As for theabove-mentioned laminin and HSPG, commercially available products can beused, and as for type IV collagen, the one extracted using acetic acidfrom bovine lens capsule can be advantageously used.

Instead of the above-mentioned method wherein a basement membranecomponent such as laminin, type IV collagen, heparan sulfateproteoglycan (HSPG), entactin and the like, or TGF-β as mentioned aboveis used, which costs highly, a basement membrane componenthyperexpressing cells into which genes of one or more types of basementmembrane components such as laminin, type IV collagen and the like aretransfected, or the growth factor hyperexpressing cells wherein theTGF-β genes are transfected can be selectively used as the cells havingan ability to form a basement membrane and fibroblasts used for themethod for preparing a basement membrane of the present invention 1.Particularly, a basement membrane specimen having a specific functioncan be obtained using the cells biosynthesizing and secreting a singlemolecular species of a basement membrane component by the genemanipulation.

As for the specific sugar chain for the method for preparing a basementmembrane of the present invention 1, namely, the sugar chain which canlocalize a receptor having an activity to accumulate a basement membranecomponent onto the basal surface of the cells having an ability to forma basement membrane, it is preferable to use a sugar chain which canadhere the cells having an ability to form a basement membrane onto asupport structure by the bonding of the sugar chain or a part of thesugar chain and the above-mentioned receptor, particularly a sugar chainwherein the sugar chain or a part of the sugar chain bonded to thereceptor can be replaced by the above-mentioned basement membranecomponent. As for the support structure with a sugar chain of thepresent invention, it is preferable to be an integral molding bodyhaving a sugar chain, or a support structure coated with polymer havinga sugar chain. The polymer having such sugar chain can be exemplified bya polymer having a sugar chain having β-D-glucopyranosyl nonreducing endor 2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end. Further thepolymer having a sugar chain having the β-D-glucopyranosyl nonreducingend can be particularly exemplified by PV-CA, PV-Lam and the like, andthe polymer having a sugar chain having the2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end can beparticularly exemplified by a polymer macromolecule whereinoligosaccharides are introduced into a vinyl monomer such as PV-GlcNAcand the like (PV-sugar). The mixture of more than one types of thesePV-sugars can also be used as well as a single type independently, andthese PV-sugars are commercially available.

The support structure (with a sugar-chain coat) for the method forpreparing a basement membrane of the present invention 1 can beexemplified by a fibrous collagen matrix, porous PET membrane,polystyreneplate, (synthetic) elastinpolymer, bioabsorbable polymer andthe like, however, a fibrous collagen matrix is more preferable in termsof maintaining the diffusion of nutritive salts and waste products. Asfor the fibrous collagen matrix, high density matrix of collagen gelcontracted by fibroblasts can also be used. In this case, it is alsopossible to add ascorbic-2-phosphate (Asc-P) in order to enhance thesynthesis of collagen. Further, a fibrous collagen matrix, which isconstructed by leaving a neutral type I collagen solution at rest in CO₂incubator to incubate it, and air-drying a resulting polymerized gel atroom temperature, can also be used. It is preferable to use abioabsorbable polymer since it is possible to transplant whilemaintaining the basement membrane structure supported by a supportstructure. Said bio absorbable polymer can be particularly exemplifiedby polyglycolic acid, poly-L-lactic acid, L-lactic acid/glycol acidcopolymer, glycol acid/68-caprolactone copolymer, L-lacticacid/ε-caprolactone copolymer, poly-ε-caprolactone and the like.

As for the tissue model of the present invention 1, there is noparticular limitation as long as it is a tissue containing cell layerswhich can be obtained by the method for preparing a basement membrane ofthe above-mentioned present invention and a basement membrane beneaththe cell layers. For example, it can be particularly exemplified byepidermal tissue model, corneal epithelial tissue model, alveolarepithelial tissue model, respiratory epithelial tissue model, renalglomerular tissue model, hepatic parenchymal tissue model, pulmonaryarterial vascular endothelial tissue model and the like. The tissuemodel of the present invention 1 has a barrier function original to aliving body since it has a cell layer and a basement membrane structuresame as those of a living body, therefore, it can be advantageously usedparticularly for applying to pharmacological test and toxicity test ofchemical substances and the like compared to conventional artificialskin and the like which does not maintain barrier function. For example,it is possible to test the safety and toxicity of a test substanceagainst an epithelial tissue by letting a test substance present ontothe cell layer of an epithelial tissue model and measuring the electricresistance between the upper surface and the basal surface of theepithelial cells. The safety and toxicity of a test substance can beevaluated since electric resistance lowers if the test substance causeda lesion, even it is a minor one, to an epithelial tissue. It is alsopossible to test the safety and toxicity of a test substance against anepithelial tissue by letting a test substance present onto the celllayer of an epithelial tissue model, and monitoring the condition of theepithelial tissue and a basement membrane using a scanning electronmicroscope or a transmission electron microscope.

As for the test tissue kit of the present invention 1, there is noparticular limitation as long as it is a kit containing a tissue modelwhich can be obtained by the method for preparing a basement membrane ofthe present invention 1, for example, it can be exemplified by a testkit for pharmacological test, toxicity test and the like containing thetissue model structure of the present invention 1, and a tissue modelconstruction kit for pharmacological test, toxicity test and, the like.The tissue model construction kit can be particularly exemplified by akit containing the following: certain cells comprising a test tissue; anextracellular matrix such as a fibrous collagen and the like coated withPV-GlcNAc and the like wherein said cells are seeded; a standard culturemedium such as DMEM culture medium and the like for culturing theabove-mentioned cells; Matrigel, type IV collagen solution, buffersolution and the like as a supplemental component. The course of theconstruction of a test tissue using said tissue model construction kitis shown in FIG. 4.

A basement membrane specimen (rBM; reconstructed basement membrane) canbe further constructed from the tissue model of the present invention 1by removing the cells having an ability to form a basement membrane froma basement membrane. Although there is no limitation to the method forremoving the cells having an ability to form a basement membrane from abasement membrane, the method for removing epithelial cells andendothelial cells without damaging the basement membrane is preferable,and it is not preferable to remove the cells using a protease such astrypsin and the like, since the basement membrane itself will also bedegraded. The method for removing epithelial cells and endothelial cellswithout damaging the basement membrane can be exemplified by a knownmethod wherein the cells which formed a basement membrane such asalveolar epithelial cells and the like are treated with 0.18 M ofhydrogen peroxide for 10 minutes, the culture was continued for 1 daythereafter, and cells are automatically detached from the basementmembrane, however, the method for constructing a basement membranespecimen of the present invention 2 is preferable.

As for the method for constructing a basement membrane specimen of thepresent invention 2, there is no particular limitation as long as it isthe method wherein the cells having an ability to form a basementmembrane adhered onto a support structure through a basement membraneare removed using a solvent having the ability to lyse lipid of saidcells and alkaline solution, however, the method wherein the treatmentto remove proteinous and nucleic residues is conducted using alkalinesolution after or at the same time as the delipidating treatment using asolvent having the ability to lyse lipid is preferable. The basementmembrane specimen used herein means a basement membrane having afunction to control morphology, differentiation, proliferation,motility, function expression and the like of cells when a desiredhomogeneous or heterogeneous cells having an ability to form a basementmembrane are seeded and cultured, and the above-mentioned cells havingan ability to form a basement membrane can be exemplified by epithelialcells, endothelial cells, mesenchymal cells and the like. Further, theabove-mentioned epithelial cells can be more particularly exemplified byepidermal cells, corneal epithelial cells, alveolar epithelial cells,mucosal epithelial cells of digestive organ system, renal glomerularepithelial cells, hepatic parenchymal cells and the like, and theabove-mentioned endothelial cells can be more particularly exemplifiedby renal glomerular capillary endothelial cells, pulmonary arterialvascular endothelial cells, placental venous vascular endothelial cells,aortic endothelial cells etc., and the mesenchymal cells can be moreparticularly exemplified by muscle cells, adipocytes, glial cells,Schwann cells and the like. The above-mentioned cells having an abilityto form a basement membrane which are adhered onto a support structurethrough the above-mentioned basement membrane includes a tissue which isan aggregate of said cells, preferably an artificial tissue of human andthe like. As for said artificial tissue (tissue model), though there isno particular limitation as long as it is at issue containing celllayers and a basement membrane beneath them, it can be particularlyexemplified by epidermal tissue model, corneal epithelial tissue model,alveolar epithelial tissue model, respiratory epithelial tissue model,renal glomerular tissue model, hepatic parenchymal tissue model,pulmonary arterial vascular endothelial tissue model and the like.

There is no particular limitation to the solvent having the ability tolyse lipid of the above-mentioned cells, as long as it is a solventwhich can possibly lyse lipid of epithelial cells and endothelial cellssuch as a surface active agent, organic solvent and the like, however, asurface active agent such as Triton X-100, Lubrol PX, deoxycholic acid,cholic acid, Tween, emulgen and the like is preferable, and among themTriton X-100 is particularly preferable. As for the concentration in useof the solvent that has the ability to lyse lipid such as a surfaceactive agent and the like, for example, in the case of Triton X-100,0.01-1.0%, or particularly around 0.1% is preferable, although itdepends on the type of cells applied and the treatment time. There is noparticular limitation to the above-mentioned alkaline solution, as longas it is an alkaline solution which dissolves the proteins remained onthe basement membrane surface of the cells but does not dissolve theprotein on the basement membrane, however, the alkaline solution with pH8-14, or particularly, with pH 9-10 is preferable. The particularexamples of such alkaline solution include an alkaline solution such as20-50 mM of NH₃,1 mM of NaOH or the like.

Besides, in order to suppress the degradation of a basement membrane byendogenous protease activity such as protease of DNase I and the like inlysosomes being liberated when the cells are lysed, a method which isconducted in phosphate buffer wherein protease inhibitor, or preferably,protease inhibitors cocktail (PIC) is added is preferable. Further, inthe method for constructing a basement membrane specimen of the presentinvention, it is possible to conduct a pretreatment for the cells havingan ability to form a basement membrane adhered to a support structurethrough a basement membrane such as an artificial human tissue and thelike with vanadium salt such as 2 mM of Na₃VO₄ and the like in advance.If vanadium salt is used for pretreatment, cells will be easilyexfoliated from a basement membrane, however, it is necessary to washthe vanadium salt.

As for the method for preparing the cells having an ability to form abasement membrane adhered onto a support structure through a basementmembrane used in the construction of a basement membrane specimen of thepresent invention 2, such as, preferably, an artificial tissue of humanand the like, the examples include a known method for preparing anartificial tissue as well as the method for preparing a basementmembrane of the present invention 1. Such known method for preparing anartificial tissue can be eligibly exemplified by the following as shownin FIG. 1: the method for preparing an artificial tissue whereinepithelial cells are cultured on a pulmonary fibroblasts-embeddedcollagen matrix in upper well of culture insert (T2-Fgel); the methodfor preparing an artificial tissue wherein epithelial cells are culturedon fibrous collagen substratum in upper well in the presence of apulmonary fibroblasts-embedded collagen matrix in lower well(T2-fib-Fcm); the method for preparing an artificial tissue whereinepithelial cells are cultured on a fibrous collagen substratum in upperwell in the presence of Matrigel coat in lower well (T2-fib-MG); themethod for preparing an artificial tissue wherein epithelial cells arecultured on a fibrous collagen substratum in upper well in the presenceof growth factor TGF-β in upper well and lower wells (T2-fib-TGFβ). Asshown in FIG. 2, it can also be eligibly exemplified by the method forpreparing a new artificial tissue wherein endothelial cells are culturedon a pulmonary fibroblasts-embedded collagen gel in upper well ofculture insert (EC-Fgel).

Other examples of the method for preparing a basement membrane includemethods for increasing the supplied amounts of basement membranecomponents such as: the method for supplying the lacking basementmembrane component from cultured cells (Cell Struc. Func., 22, 603-614,1997); the method wherein a basement membrane component is addeddirectly to culture system (J. Cell Sci., 113: 859-868, 2000); themethod for promoting the biosynthesis and secreting amount of a basementmembrane component in the cells forming a basement membrane by addinggrowth factor to culture solution (Eur. J. Cell Biol., 78: 867-875,1999); the method wherein the basement membrane component overexpressingcells or the growth factor overexpressing cells into which the genes ofa basement membrane component or growth factor are transfected are used.

It can be further exemplified by the method for preparing an artificialtissue in the short term where in the degradation of a basement membranecomponent is suppressed and basement membrane formation is acceleratedusing a matrix metalloproteinase (MMP) inhibitor. The cells forming abasement membrane secrete a plurality of matrix metalloproteases (MMP)which degrade basement membrane components as well as biosynthesize andsecrete basement membrane components. Usually, a basement membrane isnot formed by the cells independently since the degradation of basementmembrane components by MMPs is larger than the secreted amount of thecomponents necessary for basement membrane formation, however, if MMPsactivities are suppressed, the basement membrane can be formed even withthe small amount of secreted basement membrane components since thecells can effectively use the basement membrane components which wassecreted by themselves for basement membrane formation. Such MMPinhibitors can be exemplified by the synthetic MMP inhibitors of thefollowing: genetically manipulated products of synthesized TIMP (tissueinhibitor of Metalloproteinase); GM6001 (Calbiochem); MMP-2/MMP-9inhibitor I (Calbiochem), CGS27023A(N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-pycolyl]amino)-methyl butaneamido hydrochloride) (J. Med. Chem. 1997, Vol. 40, p. 2525-2532); MMPinhibitor (p-NH2-Bz-Gly-Pro-D-Leu-D-Ala-NHOH) (FN-439) (BBRC, 1994, Vol.199, p. 1442-1446), Batimastat (BB-94) (J. Med. Chem. 1998, Vol. 41, p.1209-1217) and the like. As for the method for constructing the basementmembrane where in these MMP inhibitors are used, for example, thebasement membrane and the like can be formed by seeding the cells havingan ability to form a basement membrane such as alveolar epithelial cellsand the like onto the culture matrix such as fibrous collagen and thelike, adding MMP inhibitor in 100-1,000-fold amount of IC₅₀ (inhibitorconcentration for lowering the enzyme activity to 50%) against MMP-2which is one of MMP, and culturing for 10 to 2 weeks.

As for the basement membrane specimen of the present invention 2, thereis no particular limitation as long as it can be obtained by the methodfor constructing the basement membrane specimen of the above-mentionedpresent invention 2, and such basement membrane specimen whereinepithelial cells, endothelial cells and the like are exfoliated, and thebasement membrane is exposed, for example, the above-mentioned basementmembrane specimen comprised of the basement membrane structure formed byepithelial cells, endothelial cells and the like and a support structuresuch as collagen fiber and the like can be advantageously used for theprocess for producing a reconstituted artificial tissue of the presentinvention 3. Although the basement membrane cannot be stored in thecondition that the cells are attached, the basement membrane specimen ofthe present invention 2 comprised exclusively of non-cell composition asa result that the cells are removed has a merit of being easy to bestored, and can be used at any time and any place when needed. Moreover,the basement membrane specimen can be stored under refrigeration or infreezer without any problem.

Other aspects of the basement membrane specimen of the present inventioninclude a basement membrane specimen in a free state from a supportstructure. By directly transplanting a basement membrane specimenwithout a plastic membrane and the like, or the basement membranespecimen of the present invention which is not fixed to plastic surface,for example, a basement membrane specimen formed on collagen fiber, toan affected part, cells having an ability to form a basement membraneare proliferated on a basement membrane specimen, and tissue and thelike in the affected part can be constituted in vivo. The process forproducing the basement membrane of the present invention 4 can beadvantageously applied for the construction of the above-mentionedbasement membrane specimen without a plastic membrane and the like.

There is no particular limitation to the process for producing thereconstituted artificial tissue of the present invention 3, as long asit is a method for seeding and culturing certain cells having an abilityto form a basement membrane on a basement membrane specimen or anamorphously basement membrane components-deposited specimen. The cellshaving an ability to form a basement membrane with the different originof a basement membrane specimen or an amorphously basement membranecomponents-deposited specimen (hereinafter referred to as “the basementmembrane specimen and the like”) can also be used in the same manner asthe cells having an ability to form a basement membrane with the sameorigin as the basement membrane specimen and the like. Theabove-mentioned cells having an ability to form a basement membrane canbe exemplified by epithelial cells, endothelial cells, mesenchymal cellsand the like. The above-mentioned epithelial cells can be moreparticularly exemplified by epidermal cells, corneal epithelial cells,alveolar epithelial cells, mucosal epithelial cells of digestive organsystem, renal glomerular epithelial tissue, hepatic parenchymal cellsand the like, the above-mentioned endothelial cells can be moreparticularly exemplified by renal glomerular capillary endothelialcells, pulmonary arterial vascular endothelial cells, placental venousvascular endothelial cells, or aortic endothelial cells and the like,and the mesenchymal cells can be more particularly exemplified by musclecells, adipocytes, glial cells, Schwann cells and the like. As for theabove-mentioned reconstituted artificial tissue (tissue model),preferably are constituted artificial human tissue (human tissue model),there is no particular limitation as long as it is a tissue or an organof human and the like containing cell layers and a basement membranebeneath them. For example, it can be particularly exemplified byepidermal tissue model, corneal epithelial tissue model, alveolarepithelial tissue model, respiratory epithelial tissue model, renalglomerular tissue model, hepatic parenchymal tissue model, pulmonaryarterial vascular endothelial tissue model and the like.

As for the basement membrane specimen and the like used for the processfor producing reconstituted artificial tissue of the present invention3, there is no particular limitation as long as it is a specimen of abasement membrane or a basement membrane component amorphous deposit(hereinafter referred to as “the basement membrane and the like”) havinga function to control morphology, differentiation, proliferation,motility, function expression and the like of cells when desiredhomogeneous or heterogeneous cells having an ability to form a basementmembrane are seeded and cultured thereon. The term “the basementmembrane components-amorphous deposits specimen” used herein means theincomplete basement membrane comprised of amorphous structureirregularly containing basement membrane components and the like whereinsecreted material of the cells are deposited and accumulated beneath thecells, and which has an equal or less function to control morphology,differentiation, proliferation, motility, function expression and thelike of cells compared to the case of the above-mentioned basementmembrane when desired homogeneous or heterogeneous cells having anability to form a basement membrane are seeded and cultured thereon.There is no particular limitation to the method for constructing thebasement membrane and the like including the above-mentioned knownmethod such as to treat with hydrogen peroxide solution and the like.However, it can be particularly eligibly exemplified by the method forconstructing a basement membrane specimen of the present invention,namely a method for constructing a basement membrane specimen whereinthe cells having an ability to form a basement membrane which areadhered onto a support structure through a basement membrane are removedusing a solvent having the ability to lyse lipid of said cells and analkaline solution, preferably a method for constructing a basementmembrane specimen wherein the treatment to remove proteinous and nucleicresidues using an alkaline solution is conducted after or at the sametime as the delipidating treatment using solvent having the ability tolyse lipid.

Other aspects of the artificial tissue of the present invention 3 can beexemplified by the method for culturing the cells having an ability toform a basement membrane on the opposite two substratum surfaces of asupport structure. For example, a fibrous collagen is constructed on theboth sides of porous membrane, two types of cells having an ability toform a basement membrane such as a combination of epithelial cells andvascular endothelial cells and the like are seeded and cultured on theboth sides, then the diffusion of endogenous basement membranecomponents secreted from the cells having an ability to form a basementmembrane is prevented, and as a result, the effective utilization of abasement membrane component can be enhanced. As for the combination ofthese cells, the following combinations are considered: epithelial cellsand vascular endothelial cells; epithelial cells and epithelial cells;endothelial cells and endothelial cells; epithelial cells or endothelialcells and some mesenchymal cells, and the like.

According to the present invention 3, the above-mentioned basementmembrane specimen and the like wherein epithelial cells and endothelialcells are exfoliated, and a basement membrane and the like is exposedsuch as a basement membrane specimen comprised of a basement membranestructure formed by epithelial cells and endothelial cells and the like,and a fibrous support structure such as collagen fiber and the like canbe used for the culture of other cells (see FIG. 5). Although thebasement membranes of epithelium and of endothelium are not same, mostof their components are common, and the basement membrane formed byepithelial cells can be used to the constitution of endothelial cells.For example, human epithelial tissue or human endothelial tissue can bereconstituted simply by seeding and culturing the aimed human epithelialcells or human endothelial cells on the basement membrane specimen.Actually, it has been confirmed that pulmonary arterial vascularendothelial tissue can be constituted by seeding and culturing pulmonaryarterial vascular endothelial cells on the basement membrane formed bytype II alveolar epithelial cells. It is preferable to let interstitialcells (fibroblasts) of the aimed organs or tissues cocultured whenepithelial cells or vascular endothelial cells are seeded on thebasement membrane specimen to form a new epithelial or endothelialtissue since the basement membrane formation and maintenance in the newtissue will be smoothly conducted by the aid of the interstitial cells.

As mentioned above, the reconstitution method for epithelial cells andendothelial cells wherein a basement membrane specimen and the like isutilized have a high versatility, and for example, the basement membraneconstituted using rat alveolar epithelial cells can be used for thereconstitution of a human tissue, moreover, organs and tissuereconstituted from basement membrane derived from such rat alveolarepithelial cells are not limited to alveolus. In contrast, in the casethat epithelial tissue, endothelial tissue and the like are constitutedby forming a basement membrane through the culture of epithelial cellsand endothelial cells of respective organs, it takes much time and workto develop the culturing system corresponding to respective cells, andit incurs a lot of waste to separately prepare the basement membrane forrespective tissues. As mentioned above, however, it is possible toconstruct efficiently the reconstituted artificial tissue of the presentinvention 3 using the basement membrane specimen as a common basematerial for tissue reconstitution. Further, there is no particularlimitation to the culture vessel for the case of reconstitutingepithelial tissue, endothelial tissue and the like by utilizing thebasement membrane specimen, it can be applied to hollow fiber method aswell as culture insert method. When it is applied to an artificial bloodvessel, for example, it can avoid the development of thrombus, which isa problem in artificial blood vessel, and when applied to dialysistreatment, it can reduce the burden of patients.

As mentioned above, both of the tissue model and the organ model of thepresent invention 3 which are reconstituted from a basement membranespecimen and the like can also be advantageously applied topharmacological test and toxicity test of chemical substances since theyare equipped with barrier function original to a living body becausethey contain cell layers and a basement membrane structure with barrierfunction original to a living body in the same manner as a conventionaltissue model. For example, it is possible to test the safety andtoxicity of a test substance against an epithelial tissue by letting atest substance present onto the cell layer of an epithelial tissue modeland measuring the electric resistance between the apical surface and thebasal surface of the epithelial cells. The safety and toxicity of a testsubstance can be measured since electric resistance lowers if the testsubstance caused a lesion, even it is a minor one, to an epithelialtissue. It is also possible to test the safety and toxicity of a testsubstance against an epithelial tissue by letting a test substancepresent onto the cell layer of an epithelial tissue model, andmonitoring the condition of the epithelial tissue and a basementmembrane using a scanning electron microscope or a transmission electronmicroscope.

Moreover, the basement membrane specimen without plastic membrane andthe basement membrane specimen which is not fixed on plastic surface,for example, a tissue and an organ and the like reconstituted byutilizing the basement membrane specimen formed on fibrous collagenmatrix have much higher versatility since they can be transplanted whilemaintaining the basement membrane structure. Their application examplesinclude artificial microvessel with the inside diameter less than orequal to 3 mm, and human implantable artificial tissue and the like.They can be particularly eligibly exemplified by a tissue and an organwith which epithelial cells and endothelial cells exist contiguouslysuch as artificial glomerulus, artificial liver, artificial alveolus andthe like. As for the construction of the above-mentioned basementmembrane specimen without a plastic membrane, the process for producingthe basement membrane specimen of the present invention 4 can beadvantageously applied. For example, when tissue and organs and the likeare constituted by utilizing a basement membrane specimen formed onfibrous collagen matrix, a basement membrane specimen can be easily andmechanically exfoliated from plastic membrane and plastic surfaceaccording to need without any fear that a basement membrane specimen isexfoliated from plastic membrane during the operation after the cultureand to lose their value in use.

As for the basement membrane specimen or the artificial tissue of thepresent invention 4, there is no particular limitation as long as it isa basement membrane specimen or an artificial tissue formed on theprotein support structure temporarily adhered to plastic surface throughan adsorptive polymer by hydrophobic bonding having a hydrophobic linearcarbon skeleton and a functional group which can react with protein in amolecule. Besides, as for the process for producing the basementmembrane specimen or the artificial tissue which can be transplantedwhile maintaining the structure of a basement membrane of the presentinvention, there is no particular limitation as long as it is a methodcomprising the steps of; temporarily adhering the protein supportstructure onto plastic surface through an adsorptive polymer byhydrophobic bonding having a hydrophobic linear carbon skeleton and afunctional group which can react with protein in a molecule, and lettinga basement membrane specimen or an artificial tissue formed thereon, andphysically exfoliating the protein support structure supporting abasement membrane specimen or an artificial tissue from plastic surfacewhen desired. The above-mentioned adsorptive polymer by hydrophobicbonding is shown by the above-mentioned general formula [I] (in theformula, X denotes CH or NHCHCO, Y denotes CH or NHCR²CO, R¹ denotes H,alkyl group of C1-C3, alkoxy group of C1-C3 or aryl group of C6-C8, R²denotes H or alkyl group of C1-C3, Z denotes a functional group(reactional group) optionally bonded to each other, spacer denotes(—CH₂-)p or (—NHCHR³HCO-)q, R³ denotes H or alkyl group of C1-C3, mdenotes an integral number greater or equal to 1, n denotes an integralnumber between 100 and 20000, p and q independently denote 0 or integralnumbers 1-8, r denotes an integral number greater or equal to 1). Insuch general formula [I], as for R¹, the alkyl group of C1-C3 can beexemplified by methyl group, ethyl group, n-propyl group, isopropylgroup and the like, the alkoxy group of C1-C3 can be exemplified bymethoxy group, ethoxy group, propoxy group, isopropoxy group and thelike, and aryl group of C6-C8 can be exemplified by phenyl group, benzylgroup, phenethyl group, phenoxy group, benzyloxy group, phenethyloxygroup and the like. As for said adsorptive polymer by hydrophobicbonding, an adsorptive polymer by hydrophobic bonding having ahydrophobic linear skeleton such as polyvinyl chain, linear amino acidpolymer (polyglycine, polyalanine, polyphenylalanine, tyrosine, etc.),and its derivatives in a molecular, such as an adsorptive polymer byhydrophobic bonding and the like which can be adsorbed to plasticsurface, that has a reactive functional group (reactional group) whichcan directly react to said hydrophobic linear skeleton or react toprotein support structure through a spacer can be eligibly used. Therange of n in said general formula [I] is 100-20000, the molecularweight of adsorptive polymer by hydrophobic bonding shown by the generalformula [I] is preferable to be around 15,000-3,200,000.

As for the above-mentioned reactional group, there is no particularlimitation as long as it can react and bond to the functional group ofprotein support structure. Its examples include reactional group ofcarboxylic acid anhydride type, amino group, SH group and the like. Theabove-mentioned reactional group of carboxylic acid anhydride type canbe eligibly exemplified by maleic anhydride which bonds to functionalgroup such as N-terminal amino group, lysineε-amino group, SH group andthe like of protein. Although the above-mentioned amino group reacts tocarboxyl group of protein, it is preferable to add a peptide-condensingagent for the chemical bonding. The above-mentioned SH group reactsmainly to SH group of protein, but it also bonds to S—S bonding in SSexchange reaction in some cases. Further it is also possible to attach areversible protecting group which can be easily unset as long as theabove-mentioned hydrophobic linear skeleton and copolymer can be formedon said SH group.

Said adsorptive polymer by hydrophobic bonding having the reactionalgroup can be exemplified by a copolymer of one or more types selectedfrom: ethylene; unsaturated ether such as methyl vinyl ether, ethylvinyl ether, ethyl-1-propenyl ether and the like; or α-amino acid andthe like such as alanine, glycine, valine, leucine, isoleucine,phenylalanine, tyrosin and the like; and one or more types selectedfrom: dicarboxylic acid or acid imide such as maleic anhydride, maleicanhydride imidate and the like; amino acid including sulfur such aslysine, cystein and the like; monoamino dicarboxylic acid such asaspartic acid, gultamic acid and the like; diamino monocarboxylic acidsuch as lysine and the like. These copolymer can be copolymers whereindimmer, trimer and the like are bilaterally copolymerized, however, itis preferable to be an alternating copolymer. Further, in the case ofamino acid including sulfur, monoamino dicarboxylic acid, diaminomonocarboxylic acid and the like, these polymers have the structure withfunctional group in hydrophobic linear skeleton formed by condensation,therefore, they can be applied as an adsorptive polymer by hydrophobicbonding having functional group of the present invention 4. Among them,the adsorptive polymer by hydrophobic bonding having functional group ofthe present invention can be particularly exemplified by MMAC (methylvinyl ether/maleic anhydride copolymer) which is an alternatingcopolymer of maleic anhydride and unsaturated ether such as methyl vinylether, ethyl vinyl ether, ethyl-1-propenyl ether and the like. In thecase of MMAC and the like, linear polymer with methylene group as askeleton makes it possible to adsorb to plastic surface by hydrophobicbonding, however, if it is only with —CH₂—CH₂—skeleton, it is toohydrophobic and its affinity to water will lower, and there willpossibly be a disadvantage for reactivity as a result of microscopicallyrepelling water. Therefore, regarding the one wherein some H atoms ofmethylene group are substituted with alkoxy group of C1-C3 or aryl groupof C6-C8 as mentioned above, for example, regarding the one substitutedwith alkoxy group such as methoxy group, ethoxy group, propoxy group,isopropoxy group, and the like, phenoxy group, benzyloxy group,phenethyloxy group and the like, reaction efficiency is considered to beenhanced due to the presence of O atom. Besides, it is not preferable tosubstitute with OH group instead of alkoxy group since esther bondingwith carboxylic acid anhydride is made between the molecules. MMAC iseasier to use compared to the polymer which requires acetone and thelike since it is ethanol-soluble, moreover, it can be air-dried quicklyafter sprayed. Further, as for the MMAC concentration when used forcoating treatment of plastic surface as ethanol solution and the like, 2μg/ml-1 mg/ml, or particularly, 10-50 μg/ml is eligible, and suchcoating treatment can be repeated for 1-3 times according to the desiredlevel of temporary adhesion. Further, maleic anhydride which is areactional group of MMAC is to bond to amino acid of protein such ascollagen and the like, it can ionically bond to + electric charge ofprotein even if this maleic anhydride becomes carboxyl acid as a resultof reacting to water.

Besides, the above-mentioned adsorptive polymer by hydrophobic bondingcan adsorb to plastic surface irrespective of types or material ofplastic since they adsorb to plastic surface by hydrophobic bonding notby chemical bonding due to their hydrophobic linear skeleton. If proteinsupport structure is temporarily adhered to plastic surface through saidadsorptive polymer by hydrophobic bonding, and artificial tissue or abasement membrane specimen is formed thereon, it is possible tophysically exfoliate a protein support structure supporting anartificial tissue or a basement membrane specimen from plastic surfacewhen desired, and it becomes also possible to transplant the proteinsupport structure supporting the exfoliated artificial tissue orbasement membrane specimen while maintaining the structure of a basementmembrane. Further, the above-mentioned protein support structure can beeligibly exemplified by the one used for the present invention 1, thebasement membrane specimen formed on the protein support structure canbe eligibly exemplified by the basement membrane specimen of the presentinvention 2, and the artificial tissue (including artificial organ) ofhuman and the like formed on the protein support structure can beparticularly exemplified by the artificial tissue of the presentinvention 3, for example, particularly by an artificial tissue such asan artificial epidermal tissue, an artificial corneal epithelial tissue,an artificial alveolar epithelial tissue, an artificial respiratoryepithelial tissue, an artificial renal glomerular tissue, an artificialhepatic parenchymal tissue or an artificial pulmonary arterial vascularendothelial tissue, or, an artificial organ such as an artificial bloodvessel, an artificial lung, an artificial liver, an artificial kidney,an artificial skin, an artificial cornea and the like.

The present invention will be more particularly explained in thefollowing with reference to the examples, but the technical scope of theinvention will not be limited to these examples.

EXAMPLE 1 Epithelial Cells, Endothelial Cells and the Like Forming aBasement Membrane

As for epithelial cells, type II alveolar epithelial cells (obtainedfrom rats transfected with SV40-large T antigen genes; T2 cells) whichwere provided by Dr. A. Clement, Hôpital Armand Trousseau, Paris(Clement et al., Exp. Cell Res., 196: 198-205, 1991) were cultured inDMEM (Dulbecco's modified Eagle medium) wherein 10 mM of2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonicacid (HEPES) (pH 7.2),10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, Utah),penicillin, and streptomycin are added, in the atmospheric condition ofair 95%/CO₂5%, and used. As for endothelial cells, human pulmonaryarterial vascular endothelial cells (HPAE cells) purchased fromClonetics were cultured in culture medium of MCDB 131 alone wherein 10mM of HEPES (pH 7.2), 2% FBS, growth factor, penicillin, andstreptomycin are added, or culture medium of equal mixture of MCDB 131and DMEM, in the atmospheric condition of air 95%/CO₂5%, and used. Asfor fibroblasts, the one prepared from pulmonary fibroblasts derivedfrom male rats Jcl: Fischer 344 according to the method previouslydescribed (CELL STRUCTURE AND FUNCTION22: 603-614, 1997) and the humanpulmonary fibroblasts purchased from Clonetics were used.

EXAMPLE 2 Preparation of Fibrous Collagen Gel

Collagen gel fiber was prepared on the model of dense matrix of collagengel usually constituted by fibroblasts. Type I neutral collagen solutionin DMEM (pH 7.2) (0.3-0.5 mg/ml of type I collagen obtained from 0.42 mlof bovine dermis by acid extraction; Koken Co., Tokyo) were added to 4.3cm² of cultured fibroblast layer together with polyethyleneterephthalate ester membrane of 6-well culture plate (Becton DickinsonLabware, Franklin Lakes, N.J.), and incubated in CO₂ incubator for a fewhours-24 hours, then allowed to gelling. This gel was air-dried andcompressed at room temperature for 24-48 hours, and used as high-densitycollagen fiber (fib). As for the above-mentioned fibroblasts, pulmonaryfibroblasts derived from male rats Jcl: Fischer 344 were preparedaccording to the method previously described (CELL STRUCTURE ANDFUNCTION 22: 603-614, 1997).

EXAMPLE 3 Constitution of Tissue Model 1

The methods for forming epithelial tissues and endothelial tissueshaving a basement membrane beneath the type II alveolar epithelial cellsand vascular endothelial cell layers are shown in FIG. 1 and FIG. 2respectively. In order to constitute most basic tissue model, theabove-mentioned epithelial cells (T2) or endothelial cells (HPAEC) wereseeded directly on the collagen gel wherein fibroblasts were embedded,and cultured for 2 weeks (T2-Fgel in FIG. 1, EC-Fgel in FIG. 2).Further, in order to form epithelial tissue and endothelial tissuehaving a basement membrane using the above-mentioned fib as culturematrix, epithelial cells (T2) or endothelial cells (HPAEC) were directlyseeded on fib and cultured for 2 weeks (T2-fib-Fcm, T2-fib-MG andT2-fib-TGFβ in FIG. 1). In FIG. 1, Fcm shows the coculture with collagengel wherein fibroblasts are embedded (Fgel), MG shows the culturewherein the bottom of culture plate is coated with Matrigel 200 μl(Becton Dickinson), and TGFβ shows the culture wherein 1 ng/ml of TGFβis added.

EXAMPLE 4 Confirmation of the Presence of a Receptor in Epithelial Cells

96-well polystyrene plate (Becton Dickinson) were coated with variouskinds of PV-sugars (Seikagaku Corporation) with 10 μg/ml concentrationaccording to the protocol of the manufacturer, 1×10⁴ of type II ratalveolar epithelial cells were seeded thereon, and incubated in DMEMwherein 10 mM HEPES (pH 7.2) and 1% FBS were added in CO₂ incubator at37° C. for 24-48 hours. After the incubation, the cells were stainedwith crystal violet and the adhesiveness to various kinds of PV-sugarswas investigated by measuring the absorbence of 595 nm and setting thesame as cell number. Further, cell adhesion of cell adhesion factoragainst fibronectin (FN) and vitronectin (VN) coats were also conductedsimultaneously as a control experiment. The results are shown in FIG. 6.The abscissa axis in FIG. 6 shows various kinds of PV-sugars ofnonreducing end sugar chain (GlcNAc;2-acetoamide-2-deoxy-β-D-glucopyranosyl, Lam; β-D-glucopyranosyl-(1→3),CA; β-D-glucopyranosyl-(1→4), LA; β-D-glucopyranosyl, MA;α-D-glucopyranosyl, Man; β-D-mannopyranosyl, MEA; α-D-galactopyranosyl).FIG. 6 shows that type II alveolar epithelial cells have strongadhesiveness to: PV-GlcNAc with sugar chain having2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end; PV-CA or PV-Lamwith sugar chain having β-D-glucopyranosyl nonreducing end; PV-LA withsugar chain having β-D-galactopyranosyl nonreducing end. These resultsshow that type II alveolar epithelial cells express a receptor againstthese sugar chains on their basal surface.

EXAMPLE 5 Constitution of Tissue Model 2; Preparation of a BasementMembrane by Type II Alveolar, Epithelial Cells

As for the preparation of a basement membrane, a culture insertcomprised of a lower well, and an upper well which can be placed in saidlower well on concentric circle and which has a PET membrane in itsbottom was used (see FIG. 1). On the PET membrane at the bottom part ofupper well, type II alveolar epithelial cells were cultured at 37° C. inthe presence of 5% CO₂ for 2 weeks on the support structure (fib*)wherein high-density collagen fiber (fib) constructed by the methodshown in Example 2 was coated with PV-GlcNAc, PV-CA or PV-Lam lysed inDMEM with concentration of 10 μg/ml. In this culture, collagen gelwherein fibroblasts are embedded (Fgel), Matrigel (MG), or TGFβ were notadded to culture system, DMEM wherein 10 mM of HEPES (pH7.2), 1% FBS,and 0.2 mM of ascorbic acid-2-phosphate (Asc-P) were added was used forthe culture solution. Transmission electron micrographs of an alveolarepithelial tissue thus formed are shown in FIG. 7, and scanning electronmicrographs of extracellular matrices beneath an exposed alveolarepithelial tissue as a result of removing type II alveolar epithelialcell layers on the surface of an alveolar epithelial tissue thus formedaccording the method shown in Example 8 (to be hereinafter described)are shown in FIG. 8.

In FIG. 7, the bold arrows show the basement membrane lamina densa, thesmall arrows show the old basement membranes which were formed at thebeginning of the culture, and a part of which is being degraded, and thearrowheads show the regions wherein a basement membrane is not formed(scale length is 1 μm) respectively. Further, in the untreatment ofPV-sugar (Cont) in FIG. 8, only the spots existing on fib (collagenfiber, outlined bold arrow) where secretion of epithelial cells areaccumulated (*) were confirmed, but with treatment of PV-GlcNAc, PV-Lamor PV-CA, a basement membrane is flatly formed (in response to the boldarrows in FIG. 7), in PV-Lam treatment, lower part of collagen fiber(outlined bold arrow) are glimpsed from the defective window of abasement membrane part of which are lost when type II alveolarepithelial cell layer is removed (scale length is 1 μm). Based on theseresults, it has been confirmed that if the culture is conducted onhigh-density collagen fibrous support structure coated with PV-GlcNAchaving 2-acetoamide-2-deoxy-β-D-glucopyranosyl nonreducing end, PV-CA orPV-Lam having β-D-glucopyranosyl nonreducing end (GlNAc-fib*, CA-fib*,Lam-fib*), an alveolar epithelial tissue wherein a basement membrane isformed beneath type II alveolar epithelial cell layer is constituted.Further, although type II alveolar cells adhere to PV-LA havingβ-D-galactopyranosyl nonreducing end (FIG. 6), a basement membrane wasnot formed (FIGS. 7 and 8). This fact shows that cell adhesiveness tosugar chain is a necessary condition but not a sufficient condition forthe formation of a basement membrane.

EXAMPLE 6 The Acceleration Effect for a Basement Membrane Formation byMatrigel

As shown by T2-fib-MG in FIG. 1, when lower well are coated withMatrigel 200 μl, and alveolar epithelial cells are cultured onhigh-density collagen fiber (fib) for 2 weeks, a basement membrane isformed beneath epithelial cells. However, if the amount of Matrigel isless than 50 μl, a basement membrane is not formed (J. Cell Sci.,113:589-868, 2000). Even in this case, if high-density collagen fiber(fib) are constructed according to the method shown in Example 2, andculture matrix (fib*) coated with PV-GlcNAc, PV-CA or PV-Lam are usedaccording to the method shown in Example 5, a basement membrane isformed beneath type II alveolar epithelial cell layer after the 10-daysculture. FIG. 9 shows the transmission electron micrographs of anepithelial tissue (left side: untreated, and PV-GlNAc, PV-CA coating)formed as a result of 10-days coculture with Matrigel 25 μl coating thebottom of culture plate on the high density collagen fiber (fib*) coatedwith various kinds of PV-sugars, and scanning electron micrographs(right side: untreated, and PV-GlNAc, PV-CA coating) of the result ofremoving alveolar epithelial cell layers according to the method shownin Example 8 (to be hereinafter described) and exposing a basementmembrane structure just beneath them to the surface (the meanings of themarks and scale are same as in Example 5). Based on these results, ithas been confirmed that even if the amount of Matrigel is insufficient,alveolar epithelial tissues wherein a basement membrane is formed can beconstituted just beneath type II alveolar epithelial cell layer usinghigh-density collagen fibrous support structure coated with PV-GlNAc orPV-CA (GlcNAc-fib*, CA-fib*).

EXAMPLE 7 Preparation of a Basement Membrane by Human Pulmonary ArterialVascular Endothelial Cells

Human pulmonary arterial vascular endothelial (HPAE) cells were culturedaccording to the method shown in FIG. 2. More particularly, the culturewas conducted in the following way: direct culture on the collagen gelwherein human fibroblasts are embedded (Fgel) (EC-Fgel); culture onhigh-density collagen fiber (fib) in the presence of Fgel (EC-fib-Fcm);coculture with 200 μl of Matrigel on fib (EC-fib-MG); culture on fib(EC-fib). After the culture, HPAE cell layer on the surface was removedaccording to the method for referential example 1 (to be hereinafterdescribed), and extracellular matrix structure beneath the cells weremonitored by scanning electron microscope (FIG. 10). Although a basementmembrane was formed in the case of EC-Fgel, existing collagen fiber wasexposed in the case of EC-fib-Fcm, EC-fib-MG, and EC-fib and a basementmembrane was not formed as in the case of T2 cells (T2-fib-Fcm,T2-fib-MG) (the outlined bold arrows show collagen fiber. * showssecretion deposited between collagen fibers. Scale length is 1 μm).Accordingly, culture system of EC-fib-Fcm was used for the culture onhigh-density collagen fibrous support structure (fib*) coated withPV-sugar as in the case of type II alveolar epithelial cells of Example5. After the culture, HPAE cell layer on the surface was removedaccording to the method for referential Example 1, extracellular matrixstructure exposed just beneath the cells was monitored by scanningelectron microscope (FIG. 11). In the case of PV-GlNAc and PV-CAcoating, formation of a basement membrane was confirmed. In the case ofPV-Lam coating, the formation of a basement membrane was incomplete. Inthe case of PV-sugar untreated (Cont), and PV-LA, PV-MA, PV-Man, PV-MEAtreatments, secretion of epithelial cells were accumulated (*) on theexisting collagen fiber (outlined bold arrows), however, a basementmembrane was not formed. Based on these results, it has been confirmedthat if fib* coated with PV-GlNAc, PV-CA (GlcNAc-fib*, CA-fib*) areused, human pulmonary arterial vascular endothelial tissue wherein abasement membrane is formed just beneath human pulmonary arterialvascular endothelial cell layer was constituted.

EXAMPLE 8 Construction of a Basement Membrane Specimen Wherein anAlveolar Epithelial Cell Layer is Removed and a Basement Membrane isExposed

As schematically shown in FIG. 5, type II alveolar epithelial cell layerwas exfoliated from tissue model (T2-fib-MG), a basement membranespecimen wherein a basement membrane is exposed was constructed, ratrespiratory epithelial cells or human pulmonary arterial vascularepithelial cells were seeded on such constructed basement membranestructure, and respiratory epithelial tissue and vascular epithelialtissue were constructed. Firstly, 2 ml of 0.1% of Triton X-100 (surfaceactive agent) in isotonic phosphate buffer (pH7.2; PBS (−)) containingprotease inhibitors cocktail (PIC, Peptide Institute, Inc., Osaka) wasused to lyse and elute the lipid components of epithelial cells of thetype II alveolar epithelial tissue on a culture insert of upper well,and simultaneously, the procedure to lyse protein residues of the cellsremained on the basement membrane surface with coexisting 50 mM of NH₃was conducted. This procedure of Triton X-100 and NH₃ treatment wasrepeated twice (proteins of a basement membrane should not be lysed),then an alveolar epithelial cell layer was exfoliated from the basementmembrane and the basement membrane specimen wherein a basement membraneis exposed was prepared, followed by another washing with PBC (−)solution containing PIC to remove surface active agent and alkalinesolution.

EXAMPLE 9 Reconstitution of Rat Respiratory Epithelial Tissue on theBasement Membrane Structure

5×10⁵ of rat respiratory epithelial cells (SPOC1, provided by Dr. PaulNettesheim of NIEHS (National Institute of Environmental Health andSciences)) in United States were seeded on the basement membranestructure constituted in Example 8, and cultured at 37° C. in thepresence of 5% CO₂ for 1 week in mixed culture medium of Ham'sF12:DMEM=1:1 wherein 10 mM of HEPES (pH 7.2) and 1% FBS were added, thusrespiratory epithelial tissues were constituted. Transmission electronmicrographs of respiratory epithelial tissue constituted on the basementmembrane structure are shown in FIG. 12. FIG. 12A shows respiratoryepithelial cells on the basement membrane structure, FIG. 12B shows thestrongly magnified boundary surface of basal surface of respiratoryepithelial cell and basement membrane structure wherein respiratoryepithelial cells recognizing the basement membrane derived from alveolarepithelial cells are connected with an anchoring filament, FIG. 12Cshows the formation of epithelial tissue by the bonding of respiratoryepithelial cells with cell-cell junction.

EXAMPLE 10 Reconstitution of Human Vascular Epithelial Tissue on theBasement Membrane Structure

5×10⁵ of human pulmonary arterial vascular endothelial cells (Clonetics)were seeded on the basement membrane structure constituted in Example 8,and cultured at 37° C. in the presence of 5% CO₂ for 2 weeks in mixedculture medium of MCDB131:DMEM=1:1 wherein 10 mM of HEPES (pH 7.2) and2% FBS were added, thus human vascular endothelial tissue wereconstituted. Transmission electron micrographs of human vascularendothelial tissue constituted on the basement membrane structure areshown in FIG. 13. FIG. 13A shows human vascular epithelial tissueconstituted as a result of coculture with fibroblasts, FIG. 13B showshuman vascular epithelial tissue constituted in the presence ofMatrigel.

EXAMPLE 11 Construction of Amorphous Deposit Specimen of BasementMembrane Component and the Like

Even in the case that a complete basement membrane was not formed byalveolar epithelial cells, basement membrane components secreted tooutside from the cells deposited as in amorphous structure beneath thecells. It is possible to prepare secreted materials specimen which hasbecome amorphous structure by selectively removing the cells, forexample, in the condition of attaching to extracellular matrix such ascollagen fiber and the like, or in the state of directly depositing onplastic surface, since the deposits comprised of extracellular matrices,growth factors and the like are used as a culture matrix. Type IIalveolar epithelial cells (T2 cells) were seeded on high-densitycollagen fiber (fib), and cultured for 1 or 2 weeks (see theabove-mentioned Example 1-3). After the culture of 1 or 2 weeks,alveolar epithelial cell layers were removed according to the methoddescribed in Example 8, and a basement membrane specimen and the likewherein in a basement membrane and the like were exposed was prepared.FIG. 14A-C are the scanning electron micrographs respectively show thefollowing: FIG. 14A shows collagen fiber (fib); FIG. 14B shows amorphousdeposit structure (deposit-fib: shown with *) wherein secreted materialsdeposited under the cells are exposed to the surface as a result ofremoving the cells by culturing under the condition that a basementmembrane is not formed; FIG. 14C shows the basement membrane structure(rBM) which is exposed to the surface as a result of removing the cellsafter the basement membrane is formed.

EXAMPLE 12 Construction of Tissue Herein Amorphous Deposit Specimen(Deposit-fib) is Used-1

Aimed tissue can be constructed by seeding and culturing epithelialcells, endothelial cells, muscle cells, adipocytes, Schwann cells andthe like on a basement membrane specimen (FIG. 14C) or amorphous depositspecimen (FIG. 14B). Although the performance and stability of thetissue formed when the cells of the aimed tissue are seeded on amorphousdeposit are generally inferior compared to the case where they areseeded on a basement membrane specimen, they are remarkably superior inthe performance when compared to the case where cells are seededdirectly on the plastic or untreated extracellular matrix. Alveolarepithelial cells were seeded on collagen fiber (fib), amorphous depositspecimen (deposit-fib), and a basement membrane structure (rBM), andcultured for a short term (3 days). FIG. 14D-F are the transmissionelectron micrographs respectively showing the following: FIG. 14D showsthe result of culture on untreated collagen fiber (fib); FIG. 14E showsthe result of culture on amorphous deposit specimen (deposit-fib); FIG.14F shows the result of culture on a basement membrane structure (rBM)In the culture on untreated collagen fiber (fib), deposits ofextracellular matrices beneath the cells (▴) is very little, and theformation of anchoring filament connecting the cells and a basementmembrane is consequentially little. In the culture on the basementmembrane structure (rBM), anchoring filament connecting basementmembrane lamina densa (▴) and the basal surface of the cells is wellformed, it can be seen that tissue is being formed as the cells arerecognizing and accepting the basement membrane specimen as its ownbasement membrane. In the culture on amorphous deposit specimen(deposit-fib), as the deposit (▴) and the basal surface of the cells arepartially connected by an anchoring filament, tissue formation is alsoincomplete compared to the case of the basement membrane structure(rBM), however, the formation of anchoring filament is furtherprogressed compared to the case of untreated collagen fiber (fib).

EXAMPLE 13 Construction of Tissue Wherein Amorphous Deposit Specimen(Deposit-fib) is Used-2

Respiratory epithelial cells SPOC1 were seeded on collagen fiber (fib),amorphous deposit specimen (deposit-fib), and basement membranestructure (rBM), and cultured for a short term (3 days). FIG. 15A-C arethe transmission electron micrographs respectively showing thefollowing: FIG. 15A shows the result of culture on untreated collagenfiber (fib); FIG. 15B shows the result of culture on amorphous depositspecimen (deposit-fib); FIG. 15C shows the result of culture on thebasement membrane structure (rBM). In FIG. 15, ▴ shows the places wherea complete basement membrane was formed, Δ shows the places where abasement membrane was not formed. As in Example 12, in the culture onuntreated collagen fiber (fib), deposits of extracellular matricesbeneath the cells (▴) is very little, and the formation of anchoringfilament connecting the cells and a basement membrane is consequentiallylittle. In the culture on the basement membrane structure (rBM), ananchoring filament connecting the basement membrane lamina densa (▴) andthe basal surface of the cells is well formed, it can be seen thattissue is being formed as the respiratory epithelial cells arerecognizing and accepting the basement membrane structure (rBM)constructed by alveolar epithelial cells as their own basement membrane,though it is not the basement membrane formed by themselves. In theculture on amorphous deposit specimen (deposit-fib), as the deposit (▴)and the basal surface of the cells are connected by an anchoringfilament as in the case of a basement membrane specimen, although tissueformation is slightly inferior compared to the case of the basementmembrane structure (rBM), the formation of the basement membranestructure and anchoring filament is further progressed compared to thecase of untreated collagen fiber (fib).

EXAMPLE 14 Construction of a Basement Membrane According to the MMPInhibition Method

Alveolar epithelial cells were cultured on collagen fiber (fib) with theonly addition of 1% fetal bovine serum and synthetic inhibitor of matrixmetalloproteinase (MMP) and without the addition of basement membranecomponents for 2 weeks. As MMP inhibitor, GM6001 (Calbiochem) andMMP-2/MMP-9 inhibitor I (Calbiochem) were used. 1.1 μM of GM6001 and 310μM of MMP-2/MMP-9 inhibitor I (2/9 inhibitor) were added respectively.The results are shown in FIG. 16. As FIG. 16 shows, complete basementmembrane structure (▴) was formed beneath the cells by the addition ofsynthetic MMP inhibitor. In FIG. 16, A shows the places where a basementmembrane was not formed (Control).

EXAMPLE 15 Constitution of an Artificial Tissue Exfoliated from PlasticSurface Wherein the Basement Membrane Structure is Maintained

As for the adsorptive polymer by hydrophobic bonding having reactionalgroup, alternating copolymer MMAC of methyl vinyl ether and maleicanhydride was used. MMAC was lysed in 99.5% ethanol, and MMAC solutionwith the concentration of 10 μg/ml was obtained. 0.5 ml of this MMACsolution was poured into the culture insert for 6-well culture plate(Becton Dickinson), it was air-dried after the excess solution wasremoved. This procedure was repeated for 1-3 times according to thedesired extent of the temporary adhesion, then the PET membrane surfaceof the bottom of the culture insert was coated with MMAC. In the nextplace, 0.72 ml of the collagen solution containing the pulmonaryfibroblasts or the solution of collagen only prepared in Example 2 wasplaced on the above-mentioned surface-coated PET membrane, and incubatedin CO₂ incubator for 1 hour, or a few hours-24 hours, then pulmonaryfibroblasts matrix substratum (Fgel) or high-density collagen fiber(fib) was temporarily adhered to the PET membrane surface. On thisfibrous collagen substratum, type II alveolar epithelial cells (T2) wereseeded and cultured for 2 weeks as in the same manner as Example 3(T2-Fgel, or, T2-fib-FcmorT2-fib-MG), and artificial alveolar epithelialtissue was constituted. As a result of mechanically exfoliating thisartificial alveolar epithelial tissue from the PET membrane using aspatula, the artificial alveolar epithelial tissue wherein the basementmembrane structure was maintained was obtained.

INDUSTRIAL APPLICABILITY

According to the method for preparing a basement membrane of the presentinvention 1, epithelial tissue model or endothelial tissues model havinga basement membrane structure can be constituted in the moderatecondition by using a polymer having specific sugar chain, and it hasbecome possible for the basement membrane structure prepared in suchmethod to form the epithelial tissue and endothelial tissue of othertissues. These basement membrane structure and epithelialtissue/endothelial tissue can be utilized for the purpose ofmedical/biological study, for the purpose of transplantation/therapy asan artificial blood vessel, an artificial lung, an artificial liver, anartificial kidney, an artificial skin, an artificial cornea and thelike, and for the purpose of pharmacological test and toxicity test.

According to the method for constructing a basement membrane specimen ofthe present invention 2, a basement membrane specimen having functionsto control morphology, differentiation, proliferation, motility,function expression and the like of cells can be obtained when thecertain homogeneous or heterogeneous cells having an ability to form abasement membrane are seeded and cultured, and epithelial tissue modelor endothelial tissues model having a basement membrane structure can bereconstituted in the moderate condition using said basement membranespecimen, and it becomes possible for the basement membrane structureprepared in such method to form epithelial tissues and endothelialtissues of other tissues. These basement membrane structure andepithelial tissue/endothelial tissue can be utilized for the purpose ofmedical/biological study, for the purpose of transplantation/therapy asan artificial blood vessel, an artificial lung, an artificial liver, anartificial kidney, an artificial skin, an artificial cornea and thelike, and for the purpose of pharmacological test and toxicity test.

According to the process for producing reconstituted artificial tissueof the present invention 3, it is possible to produce the desiredartificial tissue easily and efficiently in the short term at any timeand any place when needed using the specimen such as the basementmembrane and the like having functions to control morphology,differentiation, proliferation, motility, function expression and thelike of cells as a common basic material for tissue constitution, and byseeding and culturing certain homogeneous or heterogeneous cells to saidcells which have formed said basement membrane. Further, thereconstituted artificial tissue of the present invention 3 having thecell layer and basement membrane equipped with the barrier functionoriginal to the living body such as an artificial blood vessel, anartificial lung, an artificial liver, an artificial kidney, anartificial skin, an artificial cornea and the like, which are obtainedby the process for producing such reconstituted artificial tissue havingversatility can be used for the purpose of medical/biological study,transplantation/therapy, and pharmacological test and toxicity test.

According to the process for producing a basement membrane specimen oran artificial tissue which can be transplanted while maintaining thebasement membrane structure of the present invention 4, the proteinsupport structure supporting an artificial tissue or a basement membranespecimen which is adhered to plastic surface during the construction ofa basement membrane or an artificial tissue, but which can be physicallyexfoliated from plastic surface when desired, and such exfoliatedprotein support structure supporting an artificial tissue or a basementmembrane specimen will be able to be transplanted while maintaining thebasement membrane structure, and be useful as a material forregenerative medicine such as an artificial blood vessel, an artificiallung, an artificial liver, an artificial kidney, an artificial skin, anartificial cornea and the like.

The invention will now be further described by the following numberedparagraphs:

-   1. A method for preparing a basement membrane wherein cells having    an ability to form a basement membrane are cultured on a support    structure with a sugar-chain coat which can localize a receptor    having an activity to accumulate basement membrane components onto a    basal surface of the cells having an ability to form a basement    membrane.-   2. The method for preparing a basement membrane according to    paragraph 1, wherein the cells having an ability to form a basement    membrane are cultured on of a support structure with both opposite    surfaces coated by a sugar chain.-   3. The method for preparing a basement membrane according to    paragraph 1 or 2, wherein a component secreted from the cells having    an ability to form a basement membrane is used as a basement    membrane component.-   4. The method for preparing a basement membrane according to any of    paragraphs 1-3, wherein a sugar-chain coat, which can possibly    adhere the cells having an ability to form a basement membrane onto    a support structure through the binding between a sugar chain or a    part of a sugar chain and a receptor, is used.-   5. The method for preparing a basement membrane according to    paragraph 4, wherein a sugar-chain coat is used, the sugar chain or    a part of the sugar chain that binds to a receptor can be replaced    by a basement membrane component.-   6. The method for preparing a basement membrane according to any of    paragraphs 1-5, wherein the support structure with a sugar-chain    coat is a support structure coated with a polymer having a sugar    chain.-   7. The method for preparing a basement membrane according to    paragraph 6, wherein the polymer having a sugar chain is a polymer    having a sugar chain with β-D-glucopyranosyl nonreducing end or    2-acetoamide-2-deoxy-β-glucopyranosyl nonreducing end.-   8. The method for preparing a basement membrane according to    paragraph 7, wherein one or more types of polymers selected from    PV-GlNAc, PV-CA and PV-Lam is used as the polymer having a sugar    chain.-   9. The method for preparing a basement membrane according to any of    paragraphs 1-8, wherein the cells having an ability to form a    basement membrane are cocultured with fibroblasts or their    alternatives.-   10. The method for preparing a basement membrane according to any of    paragraphs 1-9, wherein the cells having an ability to form a    basement membrane are cultured in the presence of one or more types    of basement membrane components.-   11. The method for preparing a basement membrane according to any of    paragraphs 1-10, wherein the cells having an ability to form a    basement membrane are cultured in the presence of TGF-β    (transforming growth factor).-   12. The method for preparing a basement membrane according to any of    paragraphs 1-11, wherein the cells having an ability to form a    basement membrane are epithelial cells, endothelial cells or    mesenchymal cells.-   13. The method for preparing a basement membrane according to any of    paragraphs 1-12, wherein the cells and/or fibroblasts having an    ability to form a basement membrane are basement membrane    component-hyperexpressing cells into which genes of one or more    types of a basement membrane component are transfected.-   14. The method for preparing a basement membrane according to any of    paragraphs 1-13, wherein the support structure is a fibrous    collagen.-   15. A tissue model which can be obtained by the method for preparing    a basement membrane according to any of paragraphs 1-14.-   16. A test tissue kit including a tissue model which can be obtained    by the method for preparing a basement membrane according to any of    paragraphs 1-14.-   17. A method for constructing a basement membrane specimen wherein    cells having an ability to form a basement membrane adhered onto a    support structure through a basement membrane are removed using a    solvent having the ability to lyse lipid of the cells and an    alkaline solution.-   18. The method for constructing a basement membrane specimen    according to paragraph 17, wherein the treatment to remove    proteinous and nucleic residues using an alkaline solution is    conducted after or at the same time as the delipidating treatment    using a solvent having the ability to lyse lipid is conducted.-   19. The method for constructing a basement membrane specimen    according to paragraph 17 or 18, wherein the solvent having the    ability to lyse lipid is a surface active agent.-   20. The method for constructing a basement membrane specimen    according to paragraph 19, wherein the surface active agent is    Triton X-100.-   21. The method for constructing a basement membrane specimen    according to any of paragraphs 17-20, wherein the alkaline solution    is an alkaline solution with pH 8-14.-   22. The method for constructing a basement membrane specimen    according to paragraph 21, wherein the alkaline solution is an    alkaline solution with pH 9-10.-   23. The method for constructing a basement membrane specimen    according to any of paragraphs 17-22, wherein a protease inhibitor    is further used.-   24. The method for constructing a basement membrane specimen    according to any of paragraphs 17-23, wherein the basement membrane    is a basement membrane prepared by culturing the cells having an    ability to form a basement membrane on a collagen gel wherein    fibroblasts are embedded.-   25. The method for constructing a basement membrane specimen    according to any of paragraphs 17-24, wherein the basement membrane    is a basement membrane prepared by culturing the cells having an    ability to form a basement membrane on a support structure with a    sugar-chain coat which can localize a receptor having an activity to    accumulate a basement membrane component on the basal surface of the    cells having an ability to form a basement membrane.-   26. The method for constructing a basement membrane specimen    according to any of paragraphs 17-25, wherein the basement membrane    is a basement membrane prepared by culturing the cells having an    ability to form a basement membrane in the presence of a matrix    metalloproteinase.-   27. The method for constructing a basement membrane specimen    according to any of paragraphs 17-26, wherein the basement membrane    is a basement membrane prepared by culturing a basement membrane    component and/or growth factor hyperexpressing cells into which one    or more types of genes and/or growth factors of a basement membrane    component are introduced.-   28. A basement membrane specimen which can be obtained by the method    for constructing a basement membrane specimen according to any of    paragraphs 17-27.-   29. The basement membrane specimen according to paragraph 28, which    is detached from a support structure.-   30. A process for producing a reconstituted artificial tissue    wherein certain cells having an ability to form a basement membrane    are seeded and cultured on a basement membrane specimen or    amorphously basement membrane components-deposited specimen.-   31. The process for producing a reconstituted artificial tissue    according to paragraph 30, wherein the cells having an ability to    form a basement membrane have a different origin from that of a    basement membrane specimen or amorphously basement membrane    components-deposited specimen.-   32. The process for producing a reconstituted artificial tissue    according to paragraph 30 or 31, wherein the basement membrane    specimen or the amorphously basement membrane components-deposited    specimen is obtained by removing the cells having an ability to form    a basement membrane which are adhered onto a support structure    through a basement membrane or basement membrane    components-amorphous deposits using a solvent having the ability to    lyse lipid of the cells and an alkaline solution.-   33. The process for producing a reconstituted artificial tissue    according to paragraph 32, wherein the solvent having the ability to    lyse lipid of cells is a surface active agent.-   34. The process for producing a reconstituted artificial tissue    according to paragraph 32 or 33, wherein the alkaline solution is an    alkaline solution with pH 8-14.-   35. The process for producing a reconstituted artificial tissue    according to paragraph 34, wherein the alkaline solution is an    alkaline solution with pH 9-10.-   36. The process for producing a reconstituted artificial tissue    according to any of paragraphs 32-35, wherein a protease inhibitor    is further used.-   37. The process for producing a reconstituted artificial tissue    according to any of paragraphs 30-36, wherein the basement membrane    specimen or the basement membrane components-amorphous deposits    specimen is obtained from a basement membrane or a basement membrane    components-amorphous deposits prepared by culturing the cells having    an ability to form a basement membrane on a collagen gel wherein    fibroblasts are embedded.-   38. The process for producing a reconstituted artificial tissue    according to any of paragraphs 30-37, wherein the basement membrane    specimen or the basement membrane components-amorphous deposits    specimen is obtained from a basement membrane or a basement membrane    components-amorphous deposits prepared by culturing the cells having    an ability to form a basement membrane on a support structure with a    sugar chain-coat which can localize a receptor having an activity to    accumulate a basement membrane component onto the basal surface of    the cells having an ability to form a basement membrane or the    surface of the basement membrane components-amorphous deposits.-   39. The process for producing a reconstituted artificial tissue    according to any of paragraphs 30-38, wherein the basement membrane    specimen or the basement membrane components-amorphous deposits    specimen is obtained from a basement membrane or a basement membrane    components-amorphous deposits prepared by culturing the cells having    an ability to form a basement membrane in the presence of a matrix    metalloproteinase.-   40. The process for producing a reconstituted artificial tissue    according to any of paragraphs 30-39, wherein the basement membrane    specimen or the basement membrane components-amorphous deposits    specimen is obtained from a basement membrane or a basement membrane    components-amorphous deposit prepared by culturing a basement    membrane component and/or growth factor hyperexpressing cells into    which one or more types of genes and/or growth factors of basement    membrane components are transfected.-   41. A reconstituted artificial tissue which can be obtained by the    production process according to any of paragraphs 30-40.-   42. The reconstituted artificial tissue according to paragraph 41,    wherein the reconstituted artificial tissue is an artificial blood    vessel, an artificial lung, an artificial liver, an artificial    kidney, an artificial skin or an artificial cornea.-   43. The reconstituted artificial tissue according to paragraph 41 or    42, wherein the reconstituted artificial tissue is an artificial    human tissue.-   44. The reconstituted artificial tissue according to any of    paragraphs 41-43, which is detached from a support structure.-   45. A method for testing the safety and toxicity of a test substance    wherein the reconstituted artificial tissue according to any of    paragraphs 41-44 is used.-   46. A basement membrane specimen or an artificial tissue which is    formed on a protein support structure temporarily adhered to plastic    surface through an adsorptive polymer by hydrophobic bonding having    a hydrophobic linear carbon skeleton and a functional group which    can react with protein in a molecule.-   47. The basement membrane specimen or the artificial tissue    according to paragraph 46, wherein the adsorptive polymer by    hydrophobic bonding is an adsorptive polymer by hydrophobic bonding    shown by the following general formula [I]:

-    (In the formula, X denotes CH or NHCHCO, Y denotes CH or NHCR²CO,    R¹ denotes H, alkyl group of C1-C3, alkoxy group of C1-C3 or aryl    group of C6-C8, R² denotes H or alkyl group of C1-C3, Z denotes a    functional group (reactional group) optionally bonded to each other,    spacer denotes (—CH₂-)p or (—NHCHR³HCO-)q, R³ denotes H or alkyl    group of C1-C3, m denotes an integral number greater or equal to 1,    n denotes an integral number between 100 and 20000, p and q    independently denote 0 or integral numbers 1-8, r denotes an    integral number greater or equal to 1)-   48. The basement membrane specimen or the artificial tissue    according to paragraph 47, wherein the adsorptive polymer by    hydrophobic bonding shown by the general formula [I] is an    alternating copolymer of methyl vinyl ether and maleic anhydride.-   49. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-48, wherein the basement membrane    specimen is a basement membrane specimen constructed by removing the    cells having an ability to form a basement membrane adhered onto a    protein support structure through a basement membrane using a    solvent having the ability to lyse lipid of the cells and an    alkaline solution.-   50. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-49, wherein the artificial tissue    is an artificial tissue prepared by culturing the cells having an    ability to form a basement membrane on a protein support structure.-   51. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-50, wherein the artificial tissue    is an artificial tissue prepared by culturing the cells having an    ability to form a basement membrane on a protein support structure    with a sugar-chain coat which can localize a receptor having an    activity to accumulate a basement membrane component onto the basal    surface of the cells having an ability to form a basement membrane.-   52. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-51, wherein the protein support    structure is a collagen gel wherein fibroblasts are embedded.-   53. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-49, wherein the artificial tissue    is an artificial tissue prepared by culturing the cells having an    ability to form a basement membrane in the presence of a matrix    metalloproteinase.-   54. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-53, wherein the artificial tissue    is an artificial tissue prepared by culturing a basement membrane    component and/or growth factor hyperexpressing cells into which one    or more types of genes and/or growth factors of basement membrane    components are transfected.-   55. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-49, wherein the artificial tissue    is a reconstituted artificial tissue prepared by seeding and    culturing the cells having certain ability to form a basement    membrane on a basement membrane specimen.-   56. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-55, wherein the cells having an    ability to form a basement membrane are epithelial cells or    endothelial cells.-   57. The basement membrane specimen or the artificial tissue    according to any of paragraphs 46-56, wherein the artificial tissue    is an artificial epidermal tissue, an artificial corneal epithelial    tissue, an artificial alveolar epithelial tissue, an artificial    respiratory epithelial tissue, an artificial renal glomerular    tissue, an artificial hepatic parenchymal tissue or an artificial    pulmonary arterial vascular endothelial tissue, or, an artificial    blood vessel, an artificial lung, an artificial liver, an artificial    kidney, an artificial skin or an artificial cornea.-   58. A process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane wherein a protein support structure    is temporarily adhered to plastic surface through an adsorptive    polymer by hydrophobic bonding having a hydrophobic linear carbon    skeleton and a functional group which can react with protein in a    molecule, and a basement membrane specimen or an artificial tissue    is formed thereon, and a protein support structure supporting a    basement membrane specimen or an artificial tissue is physically    detached from plastic surface when desired.-   59. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to paragraph 58, wherein    the adsorptive polymer by hydrophobic bonding is an adsorptive    polymer by hydrophobic bonding shown by the following general    formula [I]:

-    (In the formula, X denotes CH or NHCHCO, Y denotes CH or NHCR²CO,    R¹ denotes H, alkyl group of C1-C3, alkoxy group of C1-C3 or aryl    group of C6-C8, R² denotes H or alkyl group of C1-C3, Z denotes a    functional group (reactional group) optionally bonded to each other,    spacer denotes (—CH₂-)p or (—NHCHR³HCO-)q, R³ denotes H or alkyl    group of C1-C3, m denotes an integral number greater or equal to 1,    n denotes an integral number between 100 and 20000, p and q    independently denote 0 or integral numbers 1-8, r denotes an    integral number greater or equal to 1).-   61. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to paragraph 59, wherein    the adsorptive polymer by hydrophobic bonding shown by the general    formula [I] is an alternating copolymer of methyl vinyl ether and    maleic anhydride.-   61. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-60, wherein the basement membrane specimen is a basement membrane    specimen constructed by removing the cells having an ability to form    a basement membrane adhered onto a protein support structure through    a basement membrane using a solvent having the ability to lyse lipid    of the cells and an alkaline solution.-   62. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-61, wherein the artificial tissue is a basement membrane prepared    by culturing the cells having an ability to form a basement membrane    on a protein support structure.-   63. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-62, wherein the artificial tissue is an artificial tissue    prepared by culturing the cells having an ability to form a basement    membrane on a protein support structure with a sugar-chain coat    which can localize a receptor having an activity to accumulate a    basement membrane component onto the basal surface of the cells    having an ability to form a basement membrane.-   64. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-63, where in the artificial tissue is a reconstituted artificial    tissue prepared by seeding and culturing the cells having a certain    ability to form a basement membrane on the basement membrane    specimen.-   65. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-64, wherein the cells having an ability to form a basement    membrane are epithelial cells or endothelial cells.-   66. The process for producing a basement membrane specimen or an    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-65, wherein the protein support structure is a collagen gel    wherein fibroblasts are embedded.-   67. The process for producing the basement membrane specimen or the    artificial tissue which can be transplanted while maintaining the    structure of a basement membrane according to any of paragraphs    58-66, wherein the artificial tissue is an artificial epidermal    tissue, an artificial corneal epithelial tissue, an artificial    alveolar epithelial tissue, an artificial respiratory epithelial    tissue, an artificial renal glomerular tissue, an artificial hepatic    parenchymal tissue or an artificial pulmonary arterial vascular    endothelial tissue, or, an artificial blood vessel, an artificial    lung, an artificial liver, an artificial kidney, an artificial skin    or an artificial cornea.

1. A method for constructing a basement membrane specimen wherein the method comprises the following steps: (a) culturing cells having an ability to form a basement membrane on a support structure coated with a sugar-chain coat which can localize a receptor having an activity to accumulate a basement membrane component on the basal surface of the cells having an ability to form a basement membrane, wherein the sugar chain coat has a β-D-glucopyranosyl non-reducing end or a 2-acetoamide-2-deoxy-β-D-glucopyranosyl non-reducing end and, wherein the cells are adhered onto the support structure through the basement membrane; (b) removing cells from the basement membrane on the support structure by subjecting the cells adhered onto the support structure through the basement membrane to a delipidating treatment using a solvent having the ability to lyse lipid of the cells; (c) conducting a treatment to remove proteinous and nucleic acid residues contained by the cells using an alkaline solution after or at the same time as the delipidating treatment, whereby the cells are removed and the basement membrane is exposed on the support structure.
 2. The method for constructing a basement membrane specimen according to claim 1, wherein the solvent having the ability to lyse lipid in step (b) is a surface active agent.
 3. The method for constructing a basement membrane specimen according to claim 2, wherein the surface active agent is Triton X-100.
 4. The method for constructing a basement membrane specimen according to claim 1, wherein the alkaline solution in step (c) is an alkaline solution with pH 8-14.
 5. The method for constructing a basement membrane specimen according to claim 4, wherein the alkaline solution is an alkaline solution with pH 9-10.
 6. The method for constructing a basement membrane specimen according to claim 1, wherein the support structure is a collagen gel containing embedded fibroblasts.
 7. The method for constructing a basement membrane specimen according to claim 1, wherein the cells have an ability to form the basement membrane in the presence of a matrix metalloproteinase inhibitor.
 8. The method for constructing a basement membrane specimen according to claim 1, wherein the cells are hyperexpressing cells. 